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High diversity of small organic N observed in soil water
Soil Biology & Biochemistry 57 (2013) 444e450
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High diversity of small organic N observed in soil water Charles R. Warren* School of Biological Sciences, The University of Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 June 2012 Received in revised form 19 September 2012 Accepted 21 September 2012 Available online 19 October 2012
The pool of organic N molecules in the soil solution reflects the activity of plants, microbes and other biological processes, and thus is likely to provide information important for ecosystem N and C cycling (e.g. organic N uptake by plants). Amino acids in soil water have often been a target of study, but few previous studies have attempted to examine a broader range of organic N molecules. The aim of this study was to develop a capillary electrophoresis-mass spectrometry (CE-MS) procedure for profiling of those small (<250 Da) organic N molecules in soil water that are amenable to analysis by CE-MS (viz., cationic at low pH, ionisable by electrospray). Centrifugal extracts of soil water from a sub-alpine grassland contained approximately 100 non-redundant peaks of small organic cations, 58 of which have been identified. Consistent with earlier studies, protein amino acids and common non-protein amino acids were among the most abundant compounds. Soil water also contained large amounts of several quaternary ammonium compounds (e.g. carnitine, acetyl carnitine, betaine, choline, ergothioneine) with the pool of quaternary ammonium compounds approximately 25% of the size of the pool of common amino acids. The large amounts of quaternary ammonium compounds in soil probably reflects their dual roles in central metabolism and osmoprotection in plants and microbes. Other identified compounds included unusual amino acids (e.g. b-alanine, pipecolic acid), heterocyclic compounds derived from aromatic amino acids (e.g. 4-(hydroxymethyl)imidazole, urocanate, nicotinic acid), amines (ethanolamine, spermine), sugar amines (glucosamine), and additional putative osmolytes of microbial or plant origin (trimethylamine N oxide, ectoine). Results of this study indicate that the pool of small organic N in soil water is more diverse than generally appreciated and not necessarily dominated by protein amino acids and common non-protein amino acids. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Organic N Amino acid Quaternary ammonium Soil water Unbiased profiling
1. Introduction The profile and concentration of organic N molecules in the soil solution reflects biological processes such as uptake and efflux of organic molecules by plant roots and free-living and symbiotic microbes, activity of extracellular enzymes, and inputs of organic molecules from above. Stresses such as water deficits and freezethaw cycles that have strong effects on physiology and composition of the microbial community (Schimel et al., 2007) ought to also affect the composition of organic metabolites in soil water. Metabolites are a direct reflection of changes in physiology (Bino et al., 2004; Patti et al., 2012), the thus the soil “meta-metabolome” should be a more direct way to examine soil processes (e.g. ecosystem N and C cycling) than genes or proteins. In recent times the profile of organic N molecules in soil solution has become of particular interest with demonstration that plants can directly take up small organic forms of N such as amino acids and small peptides * Tel.: þ61 2 9351 2678; fax: þ61 2 9351 4119. E-mail address: [email protected]. 0038-0717/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.soilbio.2012.09.025
(Chapin et al., 1993; Jones and Darrah, 1993; Warren, 2006; Hill et al., 2011; Soper et al., 2011). A major impediment to use of the soil “meta-metabolome” as an indicator of soil function and interpretation of studies on organic N uptake is that we know little about the molecular nature of small organic N molecules in soil water. Previous studies of organic N in soil water have generally quantified the 20 protein amino acids and a few common non-protein amino acids (e.g. GABA) (e.g. Ivarson and Sowden, 1969; Read and Bajwa, 1985; Abuarghub and Read, 1988; Kielland, 1995; Streeter et al., 2000; Werdin-Pfisterer et al., 2009). Broader exploration of the pool of organic N in soil water has not been attempted, yet it is probable that heterocyclics and non-amino forms of N would occur also in soil water given their occurrence in bulk soil and digested/hydrolsyed soil (e.g. Schulten and Schnitzer, 1997; He et al., 2006). The comprehensive instrumental analysis of organic N molecules in soil is challenging because soil water is likely to contain the 20 protein amino acids, various non-protein amino acids, amines, amino sugars, peptides, and heterocyclics. The huge range in polarity precludes common LC separation modes such as reversed phase,
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while for GC many organic N molecules cannot be derivatized (e.g. due to steric hindrance) or are degraded during passage through the hot injection port and column or have too low a vapour pressure (Kaspar et al., 2009). CE in combination with electrospray ionization and mass spectrometry (hereafter referred to as CE-MS) is a useful analytical platform for profiling charged molecules (Harada and Fukusaki, 2009) because it can separate molecules of widely varying polarity while also resolving isomeric and isobaric ions (Chalcraft and Britz-McKibbin, 2009). CE-MS using a low pH electrolyte is a selective and comprehensive method for organic N molecules because it separates those molecules that are cations at low pH, which in the case of organic molecules means molecules containing N (or less commonly other elements such as S or Cl). At the time of writing there was only one report of CE-MS being used for analysis of organic N molecules in soil water (Oikawa et al., 2011), but the focus of the study was development of desalting procedures. The aim of this work was to develop an analytical procedure for obtaining a more complete characterization of the small molecules contributing to organic N in soil water. Fuller characterization of organic N in soil water may provide evidence for processes contributing to the pool, and help guide choice of organic N molecules for use in studies of ecosystem N cycling and N uptake by plants. A CE-MS procedure was developed and subsequently used to characterize organic N in soil water from a previously described sub-alpine grassland soil (Warren and Taranto, 2010). The aim of this work was not to identify peptides and larger molecules, and thus separation and mass spectrometer parameters were optimized for molecules <250 Da. Soil water was reasoned to be a useful sample for identifying what might be bio-available to plants and microbes because unlike disruptive procedures such as acid hydrolysis or chemical extraction of soil it does not lead to contamination of samples by metabolites released from the microbial biomass (e.g. by mechanical damage, osmotic shock, cell lysis, etc.) (Roberts and Jones, 2008). 2. Materials and methods 2.1. Soil sampling and extraction of the soil solution The soil was a humic umbrosol (World Reference Base) from a sub-alpine grassland in the Snowy Mountains of Australia that has been described previously (Warren and Taranto, 2010; Warren and Taranto, 2011). The soil was derived from Silurian Mowomba granodiorite (approximately 433 1.5 million years old). From 0 to 30 cm the soil was a well-drained sandy loam without coarse fragments >2 mm. Below 30 cm there were abundant coarse fragments of granodiorite. In the upper 30 cm, pH (H2O) was 4.5, organic C (Walkley and Black) was 12e17%, and total N was 0.2 to 0.3%. Five replicate soil samples (0e15 cm depth) were collected in late Summer/early Autumn when soils were moist (volumetric water content at 0e15 cm ¼ 20%). For the month preceding soil collection the monthly mean air temperature was 12 C and the monthly mean soil temperature was 14 C. Samples were kept at 4 C during transport and storage. Within two days of collection, samples of soil water (the “soil solution”) were extracted from soil by centrifugation. Prior to use centrifugal filter units (20-mL volume, 3 kDa molecular weight cut-off, Vivaspin 20, Sartorius, Goettingen, Germany) were rinsed by centrifuging with 3 10-mL aliquots of ultra-pure water. Moist soil of weight 15e20 g was packed into the centrifugal filter unit and soil solution was extracted by centrifuging at 3220 g for 60 minutes at 20 C. Generally 1e1.5 mL of soil solution was collected from 20 mL of soil. Soil solution was transferred to a 2-mL microfuge tube (Safe-Lock, Eppendorf, Hamburg, Germany) and then stored at 4 C until analysis. Blanks comprising 2.0 mL of ultra-pure water were carried through the same procedure.
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2.2. Chemicals Methanol, acetonitrile and formic acid were LC/MS (Optima) grade from Fisher Chemical (Scoresby, Vic, Australia). Ammonium formate (Acros Organics) and ammonium hydroxide (28e30% NH3, Sigma, Sydney, Australia) were analytical grade. All electrolytes, rinsing solutions, standards and samples were prepared with 18.2 MU ultra-pure water (Arium, Sartorius). The electrolyte of 2 M formic acid with 20% (v/v) methanol, and sheath liquid of 50% (v/v) methanol with 0.1% (v/v) formic acid were prepared fresh every week. Before use the electrolyte and sheath liquid were degassed by ultrasonicating for 10 min. A stock solution of 200 mM ammonium formate (pH 10, adjusted with ammonium hydroxide) in 50% (v/v) acetonitrile was prepared fresh every week and was subsequently used for sample preparation. Initial method development was based on 37 standards comprising protein amino acids, common non-protein amino acids and amines. After analyzing the first soil samples an additional 20 standards were purchased so as to confirm putative identifications. All 57 standards of small N-containing molecules were prepared from their free acids or salts purchased from Sigma. All standards of chiral amino acids and peptides were L enantiomers. Stock solutions of 1 mg mL1 were made in ultra-pure water or 0.1 M HCl and stored at 80 C. Stocks were thawed as necessary and thereafter kept at 4 C for no longer than one week. 2.3. Instrumentation and mass spectrometer conditions Capillary electrophoresis was performed with a commercially available instrument (P/ACE MDQ, BeckmaneCoulter, Fullerton, CA, USA) equipped with a bare fused silica capillary (50 mm i.d. by 90e 100 cm long). The CE was interfaced via a co-axial sheath-flow sprayer (G1607A, Agilent, Waldbronn, Germany) to an ion trap mass spectrometer (AmaZon SL, Bruker Daltonics, Bremen, Germany). Sheath liquid of 50% (v/v) methanol with 0.1% (v/v) formic acid was delivered at 4 mL min1 by a syringe pump (NE-1002X Microfluidics Syringe Pump, New Era Pump Systems, Farmingdale, NY, USA) driving a 10-mL PTFE-tipped gas tight syringe (SGE, Ringwood, Vic, Australia). Stable ESI spray was achieved under the following conditions: dry gas 4 L min1 N2 at 200 C, nebulizer 6 psi, electrospray in positive mode at 4.5 kV. Ion transmission was optimized for a target mass of 100 m/z using smart parameter setting. The ion trap was set to scan a range of 50e250 m/z in enhanced resolution mode (8100 u/s). Ion accumulation time was adjusted automatically by setting the ion trap’s ion charge control to 100,000 with a maximum accumulation time of 15 ms. Data were recorded as the average of five scans. With this scan range, scan speed, target for ion accumulation and averaging there was a minimum of 15 measurements across the narrowest CE peaks (3.5 s) and generally 20 measurements across most CE peaks. For identification of unknown peaks, MS/MS spectra were obtained by re-analyzing a sub-sample of samples under the same electrophoretic conditions. Auto MS/MS data acquisition was performed with three MS/MS precursor per MS, active exclusion after three spectra, include after 0.15 min, isolation width of 1.0 m/z, fragmentation amplitude of 0.6 V modulated by SmartFrag from 30% to 200%. MS/MS spectra were acquired in enhanced resolution mode from 30 to 250 m/z with ion charge control of 100,000 and a maximum accumulation time of 50 ms. 2.4. Sample preparation and electrophoreretic procedures Standards were prepared by mixing 25 mL of standard with 25 mL of 200 mM ammonium formate (pH 10) in 50% (v/v) acetonitrile, and 4.0 mL of an internal standard mixture (10 mg mL1 methionine
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sulfone and 3-aminopyrrolidone). Preparing standards and samples in ammonium formate with acetonitrile was done to achieve pHmediated stacking (method modified from: Hasan et al., 2010) and permit injection of large volumes and thereby improve detection limits. Samples of soil water were concentrated by evaporating under a stream of dry nitrogen gas from 1.5 or 1.0 mL to approximately 25 mL and then prepared as described for standards. Samples and standards were injected at 3 psi for 30 s. Separation was under positive polarity mode at 30 kV (CE current of 22e25 mA). Between runs the capillary was flushed with electrolyte for 10 min (50 psi).
250
200
m/z
446
150
100
50
2.5. Identification and quantification
23
20
39
24
Identification of molecules was an iterative process that took several months. The first step involved identification of “known” molecules based on comparison of migration times and [M þ H]þ with 37 authentic standards run under the same conditions on the same instrument. Only about one-third of molecules were identified in this first step. The second step involved integrating mass spectral peaks for all of the “unknowns” and, where possible, generation of a short list of candidate molecules. The list of candidate molecules was generated by searching data against a CE-MS library of [M þ H]þ and migration time for 364 cationic organic molecules (Baran et al., 2006) and online MS/MS databases Metlin (Smith et al., 2005), HMDB (Wishart et al., 2007) and MassBank (Horai et al., 2010). In the third step, 20 additional standards were purchased and analysed under the same conditions as samples so as to confirm putative identifications. For 35 standards 5-point standard curves were constructed from 0.01 mg mL1 to 5 mg mL1 using methionine sulfone as an internal standard (R2 0.998). For unidentified molecules, approximate concentrations were calculated assuming a response factor of 1. For molecules that were identified but standards were unavailable, approximate concentrations were calculated based on the response factor of a similar molecule (e.g. di-peptides were quantified based on response factor of AlaeAla). 3. Results 3.1. Performance of CE-MS method for small organic N In less than 30 min CE-MS provided good separation of standard amino acids and amines (Fig. 1). A handful of amino acids and amines co-migrated but were separated in the second m/z dimension owing to differing molecular weights (e.g. the group of amines eluting near the first internal standard, or glutamine with phenylalanine). Detection limits (at a signal-to-noise ratio of 3) were better than 0.06 mM with the exception of ethanolamine (0.12 mM) and glycine (0.13 mM). The effective detection limits for samples were some 20e30 times better owing to concentration of samples by evaporation prior to injection. 3.2. Small organic N molecules in the soil solution In samples of soil water detection of organic N molecules was slightly compromised by several large peaks arising from inorganic cations (e.g. Naþ, Kþ and other common salts) in the sample (Fig. 2). In the ion source of the mass spectrometer the inorganic cations formed mass spectral clusters easily distinguished by their regular pattern of repeating formate units, and an isotope pattern and m/z characteristic of inorganic cations. The largest peak due to saltformate clusters migrated faster than organic molecules and thus did not lead to interference, but there were several smaller salt peaks that migrated among the organic molecules and may have obscured some peaks.
Intensity (x107)
1.0 11 12
41 4243
33 32 31
25
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46
34
51
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50 6
15
9
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30 21
38 37
29
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27 8 10
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40 IS 52
35 36
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47 48
45
14 7
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14
49
28
13
5
22
1718
16
18
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24
Migration time (min) Fig. 1. CE-MS analysis of 51 standards at a concentration of 1 mg mL1. Data are represented as extracted ion electropherograms (lower panel, standards are represented as peaks on a plot of intensity versus migration time), and a survey plot (upper panel, molecules are represented as smears on a plot of m/z versus migration time with ion abundance represented by colours [red, high intensity; blue, low intensity/ background]). Standards were prepared in 100 mM ammonium formate (pH 10) with 25% acetonitrile, injected at 3 psi for 30 s, separated at 30 kV in a 100 cm long 50 mm i.d. fused silica capillary with an electrolyte of 2 M formic acid with 20% methanol, ionized by electrospray in positive mode at 4.5 kV, and detected by ion trap mass spectrometry with a scan range of 50e250 m/z. Peaks are identified by numbers: IS, internal standard 3-aminopyrollidine; 1, spermidine; 2, putresceine; 3, histamine; 4, imidazole; 5, ethanolamine; 6, 1,2-dimethylimidazole; 7, choline; 8, ornithine; 9, lysine; 10, 4-(hydroxymethyl)imidazole; 11, arginine; 12, histidine; 13, b-alanine; 14, acetylcholine; 15, phenylethylamine; 16, g-aminobutyric acid (GABA); 17, 5 aminolevulinic acid; 18, glycine; 19, tyramine; 20, carnitine; 21, diaminopimelic acid; 22, alanine; 23, acetyl carnitine; 24, ectoine; 25, AlaeAla; 26, glucosamine; 27, cystathionine; 28, serine; 29, valine; 30, nicotinic acid; 31, isoleucine; 32, pipecolic acid; 33, leucine; 34, trigonelline; 35, asparagines; 36, threonine; 37, proline; 38, methionine; 39, AlaeAlaeAla; 40, glutamine; 41, glutamic acid; 42, cystine; 43, phenylalanine; 44, aminoadipic acid; 45, tryptophan; 46, hydroxyectoine; 47, citrulline; 48, betaine; 49, aspartic acid; 50, tyrosine; 51, cysteineglutathione; IS, internal standard methionine sulfone. All chiral amino acids and peptides are L-amino acids. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Samples of soil water from a sub-alpine grassland contained well in excess of 100 peaks (Fig. 2), but not all peaks corresponded to organic molecules. Nevertheless, after accounting for saltformate clusters, a handful of peaks present in blanks, peaks from in-source fragmentation and adducts there were approximately 100 peaks corresponding to organic molecules (Table S1). Forty molecules were identified by matching MHþ and migration time with those of an authentic standard run on the same instrument, 14 molecules were identified by matching against library data, 3 were identified by de novo spectral interpretation, while 42 were unknowns not matching any readily available mass spectral database. The pool of common amino acids (i.e. protein amino acids plus citrulline, GABA, ornithine) was 4.8 mmol L1 (6.0 mmol N L1) and accounted for approximately half of the concentration of measured molecules (Table S1, Fig. 3). The next largest pool was 42 unknowns <250 Da. A pool of seven quaternary ammonium compounds
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250
1.4 1.2
γ -butyrobetaine
200
m/z
trigonelline
9
100
betaine
peptides
Concentration (μ mol L-1)
8 50
salt
Intensity (x107)
1.50 1.25 1.00 0.75 0.50
carnitine
14
16
18
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5
quaternary ammonium
4
3
common amino acids
0
Fig. 2. CE-MS extracted ion electropherograms (lower panel) and survey plots (upper panel) of a sample of soil water from a sub-alpine grassland. Soil water was extracted by centrifuging soil in a 3 kDa molecular weight cut-off filter. One milliliter of filtrate was evaporated to 25 mL and then prepared as described for standards. CE-MS conditions were as described in Fig. 1.
4. Discussion
0.2
“unusual” 6
Migration time (min)
including choline, carnitine, acetyl carnitine, betaine and ergothioneine accounted for 14% of the measured molecules. The remainder of the N-containing molecules in soil water were 14 tentatively identified peptides and 12 miscellaneous non-peptides that included “unusual” amino acids (e.g. b-alanine, pipecolic acid), heterocyclic compounds derived from aromatic amino acids (e.g. 4-(hydroxymethyl)imidazole, urocanate, nicotinic acid), amines (ethanolamine, spermine), sugar amines (glucosamine), and putative osmolytes of microbial or plant origin (trimethylamine N oxide, ectoine). In terms of individual molecules, glutamic acid was present in the largest concentration (0.99 mM), followed by glutamine (0.52 mM), carnitine (0.49 mM), serine (0.48 mM), aspartic acid (0.34 mM), alanine (0.33 mM), acetyl carnitine (0.33 mM), glycine (0.27 mM), proline (0.25 mM) and leucine (0.24 mM). Notably, quaternary ammonium compounds were the third and seventh most abundant molecules and comprised five of the 25 most abundant non-peptides. Another three of the 25 most abundant non-peptide molecules were molecules not generally reported in the context of organic N in soil water, namely, 4-(hydroxymethyl) imidazole, pipecolic acid and ethanolamine.
0.4
7
1 12
0.6 acetylcarnitine
unknowns
2
0.25
0.8
choline
10
0.00
1.0
ergothioneine
150
0.0 Try Hyp Met Lys His Orn citrulline Arg GABA Tyr Ile Phe Thr Val Asn Leu Pro Gly Ala Asp Ser Gln Glu
5
4
3
2
1
0
Fig. 3. Summary of N-containing molecules <250 Da in soil water from a sub-alpine grassland. The left panel shows the major classes of molecules, while the right panels show the molecules that make up the pool of quaternary ammonium compounds (top right) and common amino acids (lower right). Molecules in soil water are classified: common amino acids (protein amino acids plus GABA, ornithine, citrulline); 7 amino acids that are quaternary ammonium compounds; 12 miscellaneous molecules that are “unusual” because they are not generally reported in the context of N in soil water; 42 unknown molecules that have not been identified; 14 di-peptides that have been at least partially identified. Standard three-letter abbreviations are used for most amino acids. Data are means of five replicate samples. Full details of data can be found in Table S1.
Chalcraft and Britz-McKibbin, 2009), so only brief details of detection limits are given here. Detection limits were generally better than 0.06 mM which is within the range of detection limits reported for CE with laser-induced fluorescence (Warren, 2008), and GC and HPLC methods based on pre-column derivatization (Kaspar et al., 2009). To put this in perspective, concentrations of individual amino acids in soil water are often <1 mM (Table S1, and see also Jämtgård et al., 2010; Farrell et al., 2011a). Hence, the detection limits of CE-MS was adequate for samples of soil water owing to inherently good detection limits and an additional 20- to 30-fold pre-concentration of samples by evaporating 1 mL of soil water to the minimum volume of 25 mL required for operation of the autosampler.
4.1. Performance of CE-MS method for small organic N 4.2. Small organic N molecules in the soil solution The approach used for small organic N in soil water is based on generic CE-MS methods with a low pH electrolyte (e.g. Soga and Heiger, 2000; Soga et al., 2003; Baidoo et al., 2008; Chalcraft and Britz-McKibbin, 2009). Analytical performance of CE-MS with a low pH electrolyte has already been described in some detail (e.g. Soga and Heiger, 2000; Soga et al., 2003; Baidoo et al., 2008;
Broader characterization of the pool of small organic N reveals that while common amino acids are abundant, the pool of small organic N is chemically diverse and not necessarily dominated solely by common amino acids. Concentrations of common amino acids and the total pool of common amino acids were, in fact,
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similar to those reported in previous studies focusing solely on common amino acids. For example, the dominant amino acids in soil water from a sub-alpine grassland were neutral (Gln, Gly, Ser, Ala) or acidic (Glu, Asp), in broad agreement with other studies (Kielland, 1995; Turnbull et al., 1996; Jämtgård et al., 2010; Farrell et al., 2011a; Inselsbacher et al., 2011). The size of the total pool of common amino acids in the soil solution was 4.8 mmol L1 (6.0 mmol N L1), which is similar to recent reports for soil water extracted from agricultural (7.65 mmol N L1) and forest soils (3.99 mmol N L1, Inselsbacher et al., 2011), and agricultural soils (1.8e5.5 mmol L1, Jämtgård et al., 2010). Hence, the major point of difference with previous studies is that soil water was found to contain a greater diversity of organic N molecules (58 identified N-containing molecules plus another 42 unknowns, Table S1) than suggested by studies quantifying 20 or so common amino acids (Kielland, 1995; Turnbull et al., 1996; Andersson and Berggren, 2005; Jämtgård et al., 2010; Farrell et al., 2011a; Inselsbacher et al., 2011). Better detection limits than in previous studies does not offer a general explanation for the diversity of molecules reported here. Detection limits were in fact rather similar to methods used in previous studies (see Section 4.1), and some of the molecules and classes of molecules reported here for the first time were present at high concentrations. For example, eight of the 25 most abundant organic N molecules had not been previously reported, and quaternary ammonium compounds have not been reported in previous studies despite being five of the top 25 most abundant non-peptides. The large number of molecules reported here is instead due to the breadth of analysis. CE-MS with a low pH electrolyte can in principle analyse all molecules that can be ionized by electrospray and are cationic at low pH, which in common organic molecules means possession of one or more N (or other cationic elements such as S or Cl). In contrast, the most commonly used methods for small organic N in soil water involve pre-column derivatization and are highly selective and effectively blind to many molecules. For example, the derivatization reactions previously used for organic N in soil solution (e.g. HPLC derivatization with 6-aminoquinolyl-Nhydroxysuccinimidyl, or phenylisothiocyanate, or o-phthalaldehyde; GC derivatization with chloroformate or t-butyldimethylsilyl; CE derivatization with 3-(2-furoyl)quinoline-2-carboxyaldehyde) are effective for primary amines, in some cases effective for secondary amines, and in no cases effective for quaternary amines (Knapp, 1979; Toyo’oka, 1999). Hence, the absence of quaternary ammonium compounds in previous studies is most probably because they were not derivatised and thus were not detected. The profile of organic N molecules in soil reflected that soil can be an osmotically challenging environment. Samples were collected when soil was moist, but based on water content measured in previous years it is probable the soil was exposed to several dryrewet cycles during the preceding three months of summer (Warren and Taranto, 2011). When soil dries microbes and plants avoid dehydration by accumulating compatible solutes, whereas when dry soil is rewet microbes rapidly release accumulated solutes to avoid a rapid influx of water into cells, drastic increase in turgor and possible cellular rupture (Schimel et al., 2007). Not surprisingly many of the most abundant metabolites were compatible solutes (osmolytes) which can accumulate to high concentrations and not affect protein structure or disrupt enzyme activity, for example, proline, quaternary ammonium compounds (carnitine, acetyl carnitine, betaine, choline, ergothioneine), ectoine, and trimethylamine N oxide (Tanret, 1909; Audley and Tan, 1968; Lippert and Galinski, 1992; Hasegawa et al., 2000; Wood et al., 2001). In general, microbes can accumulate compatible solutes either by de novo synthesis or by uptake from the extracellular medium (e.g. soil
solution) (Wood et al., 2001). The large amounts of compatible solutes in the soil solution indicate that uptake from the soil solution could be an important pathway for microbial accumulation of compatible solutes in addition to a possible source of N for uptake by higher plants. The second largest pool of compounds was 42 molecules that could not be identified (Table S1), and unknowns were six of the 25 most abundant non-peptides. On the one hand this highlights how much remains to be learnt, but on the other hand it is not surprising: metabolomic analyses of single species commonly report more than 50% of metabolites are unknowns (Lisec et al., 2006; Sugimoto et al., 2010). Teenty-four of the unknowns have nominal masses matching those of di-peptides, and thus a maximum of 24 unknowns could be di-peptides and a minimum of 18 unknowns are non-peptides. Until such time as the unknowns can be identified, or at the very least placed in broad classes, there will be large uncertainty in the total size of non-peptide versus peptide pools. The aim of this study was not to identify peptides because the method is sub-optimal for peptides; nevertheless, 14 peptides were at least partially identified based on MS/MS spectra. This is clearly an underestimate of di-peptide diversity because at least some of the unknowns are probably di-peptides and, more importantly, the scan range of m/z 50e250 meant that it was possible to measure fewer than 50% of the possible di-peptide combinations. Direct measurement of molecular forms of peptides are timely because recent studies have suggested that the pool of small peptides (less than 1 kDa) may be as large or larger than the pool of amino acids (e.g. Farrell et al., 2011a, b). To date we know almost nothing about the molecular forms of peptides in soil water, but this information is important given that the behavior of peptides will vary depending upon number and nature of amino acid residues. For amino acids we know that acidic, neutral and basic amino acids diffuse through the soil at different rates (Owen and Jones, 2001) and by analogy the rate of diffusion of peptides will be a function of the number of acidic, neutral and basic amino acids. An extra layer of complexity with peptides is that solubility varies with the number and nature of amino acid residues such that even short peptides of five amino acids can be insoluble if all amino acids are hydrophobic. Clearly, many of the questions regarding the importance of peptides to N cycling and plant N nutrition require knowledge of the molecular nature of peptides. 4.3. Limitations Additional studies on other soils are required to establish whether the profile of small organic N molecules reported here is reflected in other soils. In this context it is worth noting that the identified molecules are products of ubiquitous microbial physiology and biochemistry (Lippert and Galinski, 1992; Hasegawa et al., 2000; Wood et al., 2001) and/or have previously been described from soil (e.g. ergothioneine, Tanret, 1909; Audley and Tan, 1968). The measurements reported here almost certainly underestimate the number of small organic N molecules for four reasons. First, measurements were made on soil at one time of the year under one set of conditions, but it is probable that the total number of molecules would increase if samples were also collected at other times of the year or under abiotic stresses. For example, during summer drought, winter frost or waterlogging one would expect large changes in profiles of organic N in the short-term due to upregulation of some metabolic pathways and downregulation of others (e.g. associated with osmoregulation, cryotolerance, oxidative vs. reductive metabolism) and in the longer-term due to changes in composition of the microbial community. Second, the analytical approach cannot analyse organic N molecules that are neutral or anionic at low pH, e.g. N-acetylglucosamine and
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nucleoside mono- di- and tri-phosphates. Third, signals from abundant molecules (e.g. glutamine, glutamate, carnitine, acetyl carnitine) may obscure weaker signals from molecules present at several orders of magnitude smaller concentrations. The final reason that organic N in soil water is more diverse than reported here is because the CE-MS method used here cannot generally separate D- and L-isomers, but one would expect soil water to contain a mixture of D- and L-isomers of amino acids and peptides. 4.4. Implications and recommendations for studies of small organic N Studies of organic N uptake and cycling generally involve addition of isotope labeled organic N molecules (Chapin et al., 1993; Jones and Darrah, 1993; Warren, 2006; Hill et al., 2011 ), and this ought to be guided by knowledge of which forms of organic N occur in soil water. For example, one logical approach would be to study the uptake and cycling of those small organic N molecules that are most abundant in soil water. Previous studies have focused on uptake and cycling of protein amino acids (Chapin et al., 1993; Jones and Darrah, 1993; Warren, 2006) presumably because protein amino acids were assumed to be the most abundant small organic N molecules. However, results of this study indicate that the pool of small organic N in soil water is more diverse and there can be large amounts of several quaternary ammonium compounds and other N-containing compatible solutes. In principle, N-containing compatible solutes could make a significant contribution to ecosystem N cycling based on large concentrations in soil water and the presence in microbes of multiple osmoregulatory transporters with overlapping substrate specificities (Wood et al., 2001), though this needs to be evaluated in future studies. 4.5. Conclusions We currently have an incomplete view of the pool of small organic N in soil water. Until now common amino acids and small peptides have assumed centre stage, but at least for the soil examined in this study the time has come for common amino acids and peptides to step aside and share centre stage with a cast of other organic N molecules such as quaternary ammonium compounds. The profile of organic N molecules in soil reflected that soil can be an osmotically challenging environment. Some of the most abundant molecules were quaternary ammonium compounds and other Ncontaining compatible solutes. In principle, N-containing compatible solutes of microbial and/or plant origin could make a significant contribution to ecosystem N cycling. The exciting next steps are to contrast the profiles of small organic N molecules in soils under a range of biotic and abiotic conditions; and determine the roles of quaternary ammonium compounds and uncommon amino acids in N cycling. Acknowledgements Charles Warren is supported by a Future Fellowship from the Australian Research Council. Dr. Matthias Pelzing is thanked for assisting with set-up of CE-MS. Dr. Maria Taranto collected the soil used in this experiment. The comments of two reviewers helped to greatly improve this manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2012.09.025.
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References Abuarghub, S.M., Read, D.J., 1988. The biology of mycorrhiza in the Ericaceae .12. Quantitative-analysis of individual free amino-acids in relation to time and depth in the soil-profile. New Phytologist 108, 433e441. Andersson, P., Berggren, D., 2005. Amino acids, total organic and inorganic nitrogen in forest floor soil solution at low and high nitrogen input. Water, Air and Soil Pollution 162, 369e384. Audley, B.G., Tan, C.H., 1968. The uptake of ergothioneine from the soil into the latex of Hevea brasiliensis. Phytochemistry 7, 1999e2000. Baidoo, E.E.K., et al., 2008. Capillary electrophoresis-Fourier transform ion cyclotron resonance mass spectrometry for the identification of cationic metabolites via a pH-mediated stacking-transient isotachophoretic method. Analytical Chemistry 80, 3112e3122. Baran, R., et al., 2006. MathDAMP: a package for differential analysis of metabolite profiles. BMC Bioinformatics 7. Bino, R.J., et al., 2004. Potential of metabolomics as a functional genomics tool. Trends in Plant Science 9, 418e425. Chalcraft, K.R., Britz-McKibbin, P., 2009. Newborn screening of inborn errors of metabolism by capillary electrophoresis-electrospray ionization-mass spectrometry: a second-tier method with improved specificity and sensitivity. Analytical Chemistry 81, 307e314. Chapin, F.S., Moilanen, L., Kielland, K., 1993. Preferential use of organic nitrogen for growth by a nonmycorrhizal Arctic sedge. Nature 361, 150e153. Farrell, M., Hill, P.W., Farrar, J., Bardgett, R.D., Jones, D.L., 2011a. Seasonal variation in soluble soil carbon and nitrogen across a grassland productivity gradient. Soil Biology & Biochemistry 43, 835e844. Farrell, M., Hill, P.W., Wanniarachchi, S.D., Farrar, J., Bardgett, R.D., Jones, D.L., 2011b. Rapid peptide metabolism: A major component of soil nitrogen cycling? Global Biogeochemical Cycles 25. Harada, K., Fukusaki, E., 2009. Profiling of primary metabolite by means of capillary electrophoresis-mass spectrometry and its application for plant science. Plant Biotechnology 26, 47e52. Hasan, M.N., Park, S.H., Oh, E., Song, E.J., Ban, E., Yoo, Y.S., 2010. Sensitivity enhancement of CE and CE-MS for the analysis of peptides by a dynamic pH junction. Journal of Separation Science 33, 3701e3709. Hasegawa, P.M., Bressan, R.A., Zhu, J.K., Bohnert, H.J., 2000. Plant cellular and molecular responses to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology 51, 463e499. He, H.B., Xie, H.T., Zhang, X.D., 2006. A novel GCMS technique to assess N-15 and C13 incorporation into soil amino sugars. Soil Biology & Biochemistry 38, 1083e 1091. Hill, P.W., et al., 2011. Acquisition and assimilation of nitrogen as peptide-bound and D-enantiomers of amino acids by wheat. Plos One 6. Horai, H., et al., 2010. MassBank: a public repository for sharing mass spectral data for life sciences. Journal of Mass Spectrometry 45, 703e714. Inselsbacher, E., Ohlund, J., Jämtgård, S., Huss-Danell, K., Näsholm, T., 2011. The potential of microdialysis to monitor organic and inorganic nitrogen compounds in soil. Soil Biology & Biochemistry 43, 1321e1332. Ivarson, K.C., Sowden, F.J., 1969. Free amino acid composition of the plant root environment under field conditions. Canadian Journal of Soil Science 49, 121e 127. Jämtgård, S., Näsholm, T., Huss-Danell, K., 2010. Nitrogen compounds in soil solutions of agricultural land. Soil Biology & Biochemistry 42, 2325e2330. Jones, D.L., Darrah, P.R., 1993. Influx and efflux of amino-acids from Zea mays L roots and their implications for N-nutrition and the rhizosphere. Plant and Soil 156, 87e90. Kaspar, H., et al., 2009. Urinary amino acid analysis: A comparison of iTRAQ (R)-LCMS/MS, GC-MS, and amino acid analyzer. Journal of Chromatography B e Analytical Technologies in the Biomedical and Life Sciences 877, 1838e1846. Kielland, K., 1995. Landscape patterns of free amino acids in arctic tundra soils. Biogeochemistry 31, 85e98. Knapp, D.R., 1979. Handbook of Analytical Derivatization Reactions. John Wiley and Sons, New York. Lippert, K., Galinski, E.A., 1992. Enzyme stabilization by ectoine-type compatible solutes e protection against heating, freezing and drying. Applied Microbiology and Biotechnology 37, 61e65. Lisec, J., Schauer, N., Kopka, J., Willmitzer, L., Fernie, A.R., 2006. Gas chromatography mass spectrometry-based metabolite profiling in plants. Nature Protocols 1, 387e396. Oikawa, A., Fujita, N., Horie, R., Saito, K., Tawaraya, K., 2011. Solid-phase extraction for metabolomic analysis of high-salinity samples by capillary electrophoresismass spectrometry. Journal of Separation Science 34, 1063e1068. Owen, A.G., Jones, D.L., 2001. Competition for amino acids between wheat roots and rhizosphere microorganisms and the role of amino acids in plant N acquisition. Soil Biology & Biochemistry 33, 651e657. Patti, G.J., Yanes, O., Siuzdak, G., 2012. Metabolomics: the apogee of the omics trilogy. Nature Reviews Molecular Cell Biology 13, 263e269. Read, D.J., Bajwa, R., 1985. Some nutritional aspects of the biology of ericaceous mycorrhizas. Proceedings of the Royal Society of Edinburgh 85B, 317e332. Roberts, P., Jones, D.L., 2008. Critical evaluation of methods for determining total protein in soil solution. Soil Biology & Biochemistry 40, 1485e1495. Schimel, J., Balser, T.C., Wallenstein, M., 2007. Microbial stress-response physiology and its implications for ecosystem function. Ecology 88, 1386e1394.
450
C.R. Warren / Soil Biology & Biochemistry 57 (2013) 444e450
Schulten, H.R., Schnitzer, M., 1997. The chemistry of soil organic nitrogen: a review. Biology and Fertility of Soils 26, 1e15. Smith, C.A., et al., 2005. METLIN e a metabolite mass spectral database. Therapeutic Drug Monitoring 27, 747e751. Soga, T., Heiger, D.N., 2000. Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry. Analytical Chemistry 72, 1236e1241. Soga, T., Ohashi, Y., Ueno, Y., Naraoka, H., Tomita, M., Nishioka, T., 2003. Quantitative metabolome analysis using capillary electrophoresis mass spectrometry. Journal of Proteome Research 2, 488e494. Soper, F.M., Paungfoo-Lonhienne, C., Brackin, R., Rentsch, D., Schmidt, S., Robinson, N., 2011. Arabidopsis and Lobelia anceps access small peptides as a nitrogen source for growth. Functional Plant Biology 38, 788e796. Streeter, T.C., Bol, R., Bardgett, R.D., 2000. Amino acids as a nitrogen source in temperate upland grasslands: the use of dual labelled (C-13, N-15) glycine to test for direct uptake by dominant grasses. Rapid Communications in Mass Spectrometry 14, 1351e1355. Sugimoto, M., Hirayama, A., Robert, M., Abe, S., Soga, T., Tomita, M., 2010. Prediction of metabolite identity from accurate mass, migration time prediction and isotopic pattern information in CE-TOFMS data. Electrophoresis 31, 2311e2318. Tanret, C., 1909. The new base drawn from rye ergot, ergothioneine. Comptes Rendus Hebdomadaires Des Seances De L Academie Des Sciences 149, 222e224.
Toyo’oka, T., 1999. Modern Derivatization Methods for Separation Sciences, England. Turnbull, M.H., Schmidt, S., Erskine, P.D., Richards, S., Stewart, G.R., 1996. Root adaptation and nitrogen source acquisition in natural ecosystems. Tree Physiology 16, 941e948. Warren, C.R., 2006. Potential organic and inorganic N uptake by six Eucalyptus species. Functional Plant Biology 33, 653e660. Warren, C.R., 2008. Rapid and sensitive quantification of amino acids in soil extracts by capillary electrophoresis with laser-induced fluorescence. Soil Biology & Biochemistry 40, 916e923. Warren, C.R., Taranto, M.T., 2010. Temporal variation in pools of amino acids, inorganic and microbial N in a temperate grassland soil. Soil Biology & Biochemistry 42, 353e359. Warren, C.R., Taranto, M.T., 2011. Ecosystem respiration in a seasonally snowcovered subalpine grassland. Arctic Antarctic and Alpine Research 43, 137e146. Werdin-Pfisterer, N.R., Kielland, K., Boone, R.D., 2009. Soil amino acid composition across a boreal forest successional sequence. Soil Biology & Biochemistry 41, 1210e1220. Wishart, D.S., et al., 2007. HMDB: the human metabolome database. Nucleic Acids Research 35, D521eD526. Wood, J.M., et al., 2001. Osmosensing and osmoregulatory compatible solute accumulation by bacteria. Comparative Biochemistry and Physiology a-Molecular and Integrative Physiology 130, 437e460.
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Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
High diversity of small organic N observed in soil water Charles R. Warren* School of Biological Sciences, The University of Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 June 2012 Received in revised form 19 September 2012 Accepted 21 September 2012 Available online 19 October 2012
The pool of organic N molecules in the soil solution reflects the activity of plants, microbes and other biological processes, and thus is likely to provide information important for ecosystem N and C cycling (e.g. organic N uptake by plants). Amino acids in soil water have often been a target of study, but few previous studies have attempted to examine a broader range of organic N molecules. The aim of this study was to develop a capillary electrophoresis-mass spectrometry (CE-MS) procedure for profiling of those small (<250 Da) organic N molecules in soil water that are amenable to analysis by CE-MS (viz., cationic at low pH, ionisable by electrospray). Centrifugal extracts of soil water from a sub-alpine grassland contained approximately 100 non-redundant peaks of small organic cations, 58 of which have been identified. Consistent with earlier studies, protein amino acids and common non-protein amino acids were among the most abundant compounds. Soil water also contained large amounts of several quaternary ammonium compounds (e.g. carnitine, acetyl carnitine, betaine, choline, ergothioneine) with the pool of quaternary ammonium compounds approximately 25% of the size of the pool of common amino acids. The large amounts of quaternary ammonium compounds in soil probably reflects their dual roles in central metabolism and osmoprotection in plants and microbes. Other identified compounds included unusual amino acids (e.g. b-alanine, pipecolic acid), heterocyclic compounds derived from aromatic amino acids (e.g. 4-(hydroxymethyl)imidazole, urocanate, nicotinic acid), amines (ethanolamine, spermine), sugar amines (glucosamine), and additional putative osmolytes of microbial or plant origin (trimethylamine N oxide, ectoine). Results of this study indicate that the pool of small organic N in soil water is more diverse than generally appreciated and not necessarily dominated by protein amino acids and common non-protein amino acids. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Organic N Amino acid Quaternary ammonium Soil water Unbiased profiling
1. Introduction The profile and concentration of organic N molecules in the soil solution reflects biological processes such as uptake and efflux of organic molecules by plant roots and free-living and symbiotic microbes, activity of extracellular enzymes, and inputs of organic molecules from above. Stresses such as water deficits and freezethaw cycles that have strong effects on physiology and composition of the microbial community (Schimel et al., 2007) ought to also affect the composition of organic metabolites in soil water. Metabolites are a direct reflection of changes in physiology (Bino et al., 2004; Patti et al., 2012), the thus the soil “meta-metabolome” should be a more direct way to examine soil processes (e.g. ecosystem N and C cycling) than genes or proteins. In recent times the profile of organic N molecules in soil solution has become of particular interest with demonstration that plants can directly take up small organic forms of N such as amino acids and small peptides * Tel.: þ61 2 9351 2678; fax: þ61 2 9351 4119. E-mail address: [email protected]. 0038-0717/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.soilbio.2012.09.025
(Chapin et al., 1993; Jones and Darrah, 1993; Warren, 2006; Hill et al., 2011; Soper et al., 2011). A major impediment to use of the soil “meta-metabolome” as an indicator of soil function and interpretation of studies on organic N uptake is that we know little about the molecular nature of small organic N molecules in soil water. Previous studies of organic N in soil water have generally quantified the 20 protein amino acids and a few common non-protein amino acids (e.g. GABA) (e.g. Ivarson and Sowden, 1969; Read and Bajwa, 1985; Abuarghub and Read, 1988; Kielland, 1995; Streeter et al., 2000; Werdin-Pfisterer et al., 2009). Broader exploration of the pool of organic N in soil water has not been attempted, yet it is probable that heterocyclics and non-amino forms of N would occur also in soil water given their occurrence in bulk soil and digested/hydrolsyed soil (e.g. Schulten and Schnitzer, 1997; He et al., 2006). The comprehensive instrumental analysis of organic N molecules in soil is challenging because soil water is likely to contain the 20 protein amino acids, various non-protein amino acids, amines, amino sugars, peptides, and heterocyclics. The huge range in polarity precludes common LC separation modes such as reversed phase,
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while for GC many organic N molecules cannot be derivatized (e.g. due to steric hindrance) or are degraded during passage through the hot injection port and column or have too low a vapour pressure (Kaspar et al., 2009). CE in combination with electrospray ionization and mass spectrometry (hereafter referred to as CE-MS) is a useful analytical platform for profiling charged molecules (Harada and Fukusaki, 2009) because it can separate molecules of widely varying polarity while also resolving isomeric and isobaric ions (Chalcraft and Britz-McKibbin, 2009). CE-MS using a low pH electrolyte is a selective and comprehensive method for organic N molecules because it separates those molecules that are cations at low pH, which in the case of organic molecules means molecules containing N (or less commonly other elements such as S or Cl). At the time of writing there was only one report of CE-MS being used for analysis of organic N molecules in soil water (Oikawa et al., 2011), but the focus of the study was development of desalting procedures. The aim of this work was to develop an analytical procedure for obtaining a more complete characterization of the small molecules contributing to organic N in soil water. Fuller characterization of organic N in soil water may provide evidence for processes contributing to the pool, and help guide choice of organic N molecules for use in studies of ecosystem N cycling and N uptake by plants. A CE-MS procedure was developed and subsequently used to characterize organic N in soil water from a previously described sub-alpine grassland soil (Warren and Taranto, 2010). The aim of this work was not to identify peptides and larger molecules, and thus separation and mass spectrometer parameters were optimized for molecules <250 Da. Soil water was reasoned to be a useful sample for identifying what might be bio-available to plants and microbes because unlike disruptive procedures such as acid hydrolysis or chemical extraction of soil it does not lead to contamination of samples by metabolites released from the microbial biomass (e.g. by mechanical damage, osmotic shock, cell lysis, etc.) (Roberts and Jones, 2008). 2. Materials and methods 2.1. Soil sampling and extraction of the soil solution The soil was a humic umbrosol (World Reference Base) from a sub-alpine grassland in the Snowy Mountains of Australia that has been described previously (Warren and Taranto, 2010; Warren and Taranto, 2011). The soil was derived from Silurian Mowomba granodiorite (approximately 433 1.5 million years old). From 0 to 30 cm the soil was a well-drained sandy loam without coarse fragments >2 mm. Below 30 cm there were abundant coarse fragments of granodiorite. In the upper 30 cm, pH (H2O) was 4.5, organic C (Walkley and Black) was 12e17%, and total N was 0.2 to 0.3%. Five replicate soil samples (0e15 cm depth) were collected in late Summer/early Autumn when soils were moist (volumetric water content at 0e15 cm ¼ 20%). For the month preceding soil collection the monthly mean air temperature was 12 C and the monthly mean soil temperature was 14 C. Samples were kept at 4 C during transport and storage. Within two days of collection, samples of soil water (the “soil solution”) were extracted from soil by centrifugation. Prior to use centrifugal filter units (20-mL volume, 3 kDa molecular weight cut-off, Vivaspin 20, Sartorius, Goettingen, Germany) were rinsed by centrifuging with 3 10-mL aliquots of ultra-pure water. Moist soil of weight 15e20 g was packed into the centrifugal filter unit and soil solution was extracted by centrifuging at 3220 g for 60 minutes at 20 C. Generally 1e1.5 mL of soil solution was collected from 20 mL of soil. Soil solution was transferred to a 2-mL microfuge tube (Safe-Lock, Eppendorf, Hamburg, Germany) and then stored at 4 C until analysis. Blanks comprising 2.0 mL of ultra-pure water were carried through the same procedure.
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2.2. Chemicals Methanol, acetonitrile and formic acid were LC/MS (Optima) grade from Fisher Chemical (Scoresby, Vic, Australia). Ammonium formate (Acros Organics) and ammonium hydroxide (28e30% NH3, Sigma, Sydney, Australia) were analytical grade. All electrolytes, rinsing solutions, standards and samples were prepared with 18.2 MU ultra-pure water (Arium, Sartorius). The electrolyte of 2 M formic acid with 20% (v/v) methanol, and sheath liquid of 50% (v/v) methanol with 0.1% (v/v) formic acid were prepared fresh every week. Before use the electrolyte and sheath liquid were degassed by ultrasonicating for 10 min. A stock solution of 200 mM ammonium formate (pH 10, adjusted with ammonium hydroxide) in 50% (v/v) acetonitrile was prepared fresh every week and was subsequently used for sample preparation. Initial method development was based on 37 standards comprising protein amino acids, common non-protein amino acids and amines. After analyzing the first soil samples an additional 20 standards were purchased so as to confirm putative identifications. All 57 standards of small N-containing molecules were prepared from their free acids or salts purchased from Sigma. All standards of chiral amino acids and peptides were L enantiomers. Stock solutions of 1 mg mL1 were made in ultra-pure water or 0.1 M HCl and stored at 80 C. Stocks were thawed as necessary and thereafter kept at 4 C for no longer than one week. 2.3. Instrumentation and mass spectrometer conditions Capillary electrophoresis was performed with a commercially available instrument (P/ACE MDQ, BeckmaneCoulter, Fullerton, CA, USA) equipped with a bare fused silica capillary (50 mm i.d. by 90e 100 cm long). The CE was interfaced via a co-axial sheath-flow sprayer (G1607A, Agilent, Waldbronn, Germany) to an ion trap mass spectrometer (AmaZon SL, Bruker Daltonics, Bremen, Germany). Sheath liquid of 50% (v/v) methanol with 0.1% (v/v) formic acid was delivered at 4 mL min1 by a syringe pump (NE-1002X Microfluidics Syringe Pump, New Era Pump Systems, Farmingdale, NY, USA) driving a 10-mL PTFE-tipped gas tight syringe (SGE, Ringwood, Vic, Australia). Stable ESI spray was achieved under the following conditions: dry gas 4 L min1 N2 at 200 C, nebulizer 6 psi, electrospray in positive mode at 4.5 kV. Ion transmission was optimized for a target mass of 100 m/z using smart parameter setting. The ion trap was set to scan a range of 50e250 m/z in enhanced resolution mode (8100 u/s). Ion accumulation time was adjusted automatically by setting the ion trap’s ion charge control to 100,000 with a maximum accumulation time of 15 ms. Data were recorded as the average of five scans. With this scan range, scan speed, target for ion accumulation and averaging there was a minimum of 15 measurements across the narrowest CE peaks (3.5 s) and generally 20 measurements across most CE peaks. For identification of unknown peaks, MS/MS spectra were obtained by re-analyzing a sub-sample of samples under the same electrophoretic conditions. Auto MS/MS data acquisition was performed with three MS/MS precursor per MS, active exclusion after three spectra, include after 0.15 min, isolation width of 1.0 m/z, fragmentation amplitude of 0.6 V modulated by SmartFrag from 30% to 200%. MS/MS spectra were acquired in enhanced resolution mode from 30 to 250 m/z with ion charge control of 100,000 and a maximum accumulation time of 50 ms. 2.4. Sample preparation and electrophoreretic procedures Standards were prepared by mixing 25 mL of standard with 25 mL of 200 mM ammonium formate (pH 10) in 50% (v/v) acetonitrile, and 4.0 mL of an internal standard mixture (10 mg mL1 methionine
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sulfone and 3-aminopyrrolidone). Preparing standards and samples in ammonium formate with acetonitrile was done to achieve pHmediated stacking (method modified from: Hasan et al., 2010) and permit injection of large volumes and thereby improve detection limits. Samples of soil water were concentrated by evaporating under a stream of dry nitrogen gas from 1.5 or 1.0 mL to approximately 25 mL and then prepared as described for standards. Samples and standards were injected at 3 psi for 30 s. Separation was under positive polarity mode at 30 kV (CE current of 22e25 mA). Between runs the capillary was flushed with electrolyte for 10 min (50 psi).
250
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446
150
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2.5. Identification and quantification
23
20
39
24
Identification of molecules was an iterative process that took several months. The first step involved identification of “known” molecules based on comparison of migration times and [M þ H]þ with 37 authentic standards run under the same conditions on the same instrument. Only about one-third of molecules were identified in this first step. The second step involved integrating mass spectral peaks for all of the “unknowns” and, where possible, generation of a short list of candidate molecules. The list of candidate molecules was generated by searching data against a CE-MS library of [M þ H]þ and migration time for 364 cationic organic molecules (Baran et al., 2006) and online MS/MS databases Metlin (Smith et al., 2005), HMDB (Wishart et al., 2007) and MassBank (Horai et al., 2010). In the third step, 20 additional standards were purchased and analysed under the same conditions as samples so as to confirm putative identifications. For 35 standards 5-point standard curves were constructed from 0.01 mg mL1 to 5 mg mL1 using methionine sulfone as an internal standard (R2 0.998). For unidentified molecules, approximate concentrations were calculated assuming a response factor of 1. For molecules that were identified but standards were unavailable, approximate concentrations were calculated based on the response factor of a similar molecule (e.g. di-peptides were quantified based on response factor of AlaeAla). 3. Results 3.1. Performance of CE-MS method for small organic N In less than 30 min CE-MS provided good separation of standard amino acids and amines (Fig. 1). A handful of amino acids and amines co-migrated but were separated in the second m/z dimension owing to differing molecular weights (e.g. the group of amines eluting near the first internal standard, or glutamine with phenylalanine). Detection limits (at a signal-to-noise ratio of 3) were better than 0.06 mM with the exception of ethanolamine (0.12 mM) and glycine (0.13 mM). The effective detection limits for samples were some 20e30 times better owing to concentration of samples by evaporation prior to injection. 3.2. Small organic N molecules in the soil solution In samples of soil water detection of organic N molecules was slightly compromised by several large peaks arising from inorganic cations (e.g. Naþ, Kþ and other common salts) in the sample (Fig. 2). In the ion source of the mass spectrometer the inorganic cations formed mass spectral clusters easily distinguished by their regular pattern of repeating formate units, and an isotope pattern and m/z characteristic of inorganic cations. The largest peak due to saltformate clusters migrated faster than organic molecules and thus did not lead to interference, but there were several smaller salt peaks that migrated among the organic molecules and may have obscured some peaks.
Intensity (x107)
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41 4243
33 32 31
25
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15
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30 21
38 37
29
0.4
27 8 10
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40 IS 52
35 36
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0.2 IS
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47 48
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14 7
12
14
49
28
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5
22
1718
16
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22
24
Migration time (min) Fig. 1. CE-MS analysis of 51 standards at a concentration of 1 mg mL1. Data are represented as extracted ion electropherograms (lower panel, standards are represented as peaks on a plot of intensity versus migration time), and a survey plot (upper panel, molecules are represented as smears on a plot of m/z versus migration time with ion abundance represented by colours [red, high intensity; blue, low intensity/ background]). Standards were prepared in 100 mM ammonium formate (pH 10) with 25% acetonitrile, injected at 3 psi for 30 s, separated at 30 kV in a 100 cm long 50 mm i.d. fused silica capillary with an electrolyte of 2 M formic acid with 20% methanol, ionized by electrospray in positive mode at 4.5 kV, and detected by ion trap mass spectrometry with a scan range of 50e250 m/z. Peaks are identified by numbers: IS, internal standard 3-aminopyrollidine; 1, spermidine; 2, putresceine; 3, histamine; 4, imidazole; 5, ethanolamine; 6, 1,2-dimethylimidazole; 7, choline; 8, ornithine; 9, lysine; 10, 4-(hydroxymethyl)imidazole; 11, arginine; 12, histidine; 13, b-alanine; 14, acetylcholine; 15, phenylethylamine; 16, g-aminobutyric acid (GABA); 17, 5 aminolevulinic acid; 18, glycine; 19, tyramine; 20, carnitine; 21, diaminopimelic acid; 22, alanine; 23, acetyl carnitine; 24, ectoine; 25, AlaeAla; 26, glucosamine; 27, cystathionine; 28, serine; 29, valine; 30, nicotinic acid; 31, isoleucine; 32, pipecolic acid; 33, leucine; 34, trigonelline; 35, asparagines; 36, threonine; 37, proline; 38, methionine; 39, AlaeAlaeAla; 40, glutamine; 41, glutamic acid; 42, cystine; 43, phenylalanine; 44, aminoadipic acid; 45, tryptophan; 46, hydroxyectoine; 47, citrulline; 48, betaine; 49, aspartic acid; 50, tyrosine; 51, cysteineglutathione; IS, internal standard methionine sulfone. All chiral amino acids and peptides are L-amino acids. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Samples of soil water from a sub-alpine grassland contained well in excess of 100 peaks (Fig. 2), but not all peaks corresponded to organic molecules. Nevertheless, after accounting for saltformate clusters, a handful of peaks present in blanks, peaks from in-source fragmentation and adducts there were approximately 100 peaks corresponding to organic molecules (Table S1). Forty molecules were identified by matching MHþ and migration time with those of an authentic standard run on the same instrument, 14 molecules were identified by matching against library data, 3 were identified by de novo spectral interpretation, while 42 were unknowns not matching any readily available mass spectral database. The pool of common amino acids (i.e. protein amino acids plus citrulline, GABA, ornithine) was 4.8 mmol L1 (6.0 mmol N L1) and accounted for approximately half of the concentration of measured molecules (Table S1, Fig. 3). The next largest pool was 42 unknowns <250 Da. A pool of seven quaternary ammonium compounds
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447
250
1.4 1.2
γ -butyrobetaine
200
m/z
trigonelline
9
100
betaine
peptides
Concentration (μ mol L-1)
8 50
salt
Intensity (x107)
1.50 1.25 1.00 0.75 0.50
carnitine
14
16
18
20
22
5
quaternary ammonium
4
3
common amino acids
0
Fig. 2. CE-MS extracted ion electropherograms (lower panel) and survey plots (upper panel) of a sample of soil water from a sub-alpine grassland. Soil water was extracted by centrifuging soil in a 3 kDa molecular weight cut-off filter. One milliliter of filtrate was evaporated to 25 mL and then prepared as described for standards. CE-MS conditions were as described in Fig. 1.
4. Discussion
0.2
“unusual” 6
Migration time (min)
including choline, carnitine, acetyl carnitine, betaine and ergothioneine accounted for 14% of the measured molecules. The remainder of the N-containing molecules in soil water were 14 tentatively identified peptides and 12 miscellaneous non-peptides that included “unusual” amino acids (e.g. b-alanine, pipecolic acid), heterocyclic compounds derived from aromatic amino acids (e.g. 4-(hydroxymethyl)imidazole, urocanate, nicotinic acid), amines (ethanolamine, spermine), sugar amines (glucosamine), and putative osmolytes of microbial or plant origin (trimethylamine N oxide, ectoine). In terms of individual molecules, glutamic acid was present in the largest concentration (0.99 mM), followed by glutamine (0.52 mM), carnitine (0.49 mM), serine (0.48 mM), aspartic acid (0.34 mM), alanine (0.33 mM), acetyl carnitine (0.33 mM), glycine (0.27 mM), proline (0.25 mM) and leucine (0.24 mM). Notably, quaternary ammonium compounds were the third and seventh most abundant molecules and comprised five of the 25 most abundant non-peptides. Another three of the 25 most abundant non-peptide molecules were molecules not generally reported in the context of organic N in soil water, namely, 4-(hydroxymethyl) imidazole, pipecolic acid and ethanolamine.
0.4
7
1 12
0.6 acetylcarnitine
unknowns
2
0.25
0.8
choline
10
0.00
1.0
ergothioneine
150
0.0 Try Hyp Met Lys His Orn citrulline Arg GABA Tyr Ile Phe Thr Val Asn Leu Pro Gly Ala Asp Ser Gln Glu
5
4
3
2
1
0
Fig. 3. Summary of N-containing molecules <250 Da in soil water from a sub-alpine grassland. The left panel shows the major classes of molecules, while the right panels show the molecules that make up the pool of quaternary ammonium compounds (top right) and common amino acids (lower right). Molecules in soil water are classified: common amino acids (protein amino acids plus GABA, ornithine, citrulline); 7 amino acids that are quaternary ammonium compounds; 12 miscellaneous molecules that are “unusual” because they are not generally reported in the context of N in soil water; 42 unknown molecules that have not been identified; 14 di-peptides that have been at least partially identified. Standard three-letter abbreviations are used for most amino acids. Data are means of five replicate samples. Full details of data can be found in Table S1.
Chalcraft and Britz-McKibbin, 2009), so only brief details of detection limits are given here. Detection limits were generally better than 0.06 mM which is within the range of detection limits reported for CE with laser-induced fluorescence (Warren, 2008), and GC and HPLC methods based on pre-column derivatization (Kaspar et al., 2009). To put this in perspective, concentrations of individual amino acids in soil water are often <1 mM (Table S1, and see also Jämtgård et al., 2010; Farrell et al., 2011a). Hence, the detection limits of CE-MS was adequate for samples of soil water owing to inherently good detection limits and an additional 20- to 30-fold pre-concentration of samples by evaporating 1 mL of soil water to the minimum volume of 25 mL required for operation of the autosampler.
4.1. Performance of CE-MS method for small organic N 4.2. Small organic N molecules in the soil solution The approach used for small organic N in soil water is based on generic CE-MS methods with a low pH electrolyte (e.g. Soga and Heiger, 2000; Soga et al., 2003; Baidoo et al., 2008; Chalcraft and Britz-McKibbin, 2009). Analytical performance of CE-MS with a low pH electrolyte has already been described in some detail (e.g. Soga and Heiger, 2000; Soga et al., 2003; Baidoo et al., 2008;
Broader characterization of the pool of small organic N reveals that while common amino acids are abundant, the pool of small organic N is chemically diverse and not necessarily dominated solely by common amino acids. Concentrations of common amino acids and the total pool of common amino acids were, in fact,
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similar to those reported in previous studies focusing solely on common amino acids. For example, the dominant amino acids in soil water from a sub-alpine grassland were neutral (Gln, Gly, Ser, Ala) or acidic (Glu, Asp), in broad agreement with other studies (Kielland, 1995; Turnbull et al., 1996; Jämtgård et al., 2010; Farrell et al., 2011a; Inselsbacher et al., 2011). The size of the total pool of common amino acids in the soil solution was 4.8 mmol L1 (6.0 mmol N L1), which is similar to recent reports for soil water extracted from agricultural (7.65 mmol N L1) and forest soils (3.99 mmol N L1, Inselsbacher et al., 2011), and agricultural soils (1.8e5.5 mmol L1, Jämtgård et al., 2010). Hence, the major point of difference with previous studies is that soil water was found to contain a greater diversity of organic N molecules (58 identified N-containing molecules plus another 42 unknowns, Table S1) than suggested by studies quantifying 20 or so common amino acids (Kielland, 1995; Turnbull et al., 1996; Andersson and Berggren, 2005; Jämtgård et al., 2010; Farrell et al., 2011a; Inselsbacher et al., 2011). Better detection limits than in previous studies does not offer a general explanation for the diversity of molecules reported here. Detection limits were in fact rather similar to methods used in previous studies (see Section 4.1), and some of the molecules and classes of molecules reported here for the first time were present at high concentrations. For example, eight of the 25 most abundant organic N molecules had not been previously reported, and quaternary ammonium compounds have not been reported in previous studies despite being five of the top 25 most abundant non-peptides. The large number of molecules reported here is instead due to the breadth of analysis. CE-MS with a low pH electrolyte can in principle analyse all molecules that can be ionized by electrospray and are cationic at low pH, which in common organic molecules means possession of one or more N (or other cationic elements such as S or Cl). In contrast, the most commonly used methods for small organic N in soil water involve pre-column derivatization and are highly selective and effectively blind to many molecules. For example, the derivatization reactions previously used for organic N in soil solution (e.g. HPLC derivatization with 6-aminoquinolyl-Nhydroxysuccinimidyl, or phenylisothiocyanate, or o-phthalaldehyde; GC derivatization with chloroformate or t-butyldimethylsilyl; CE derivatization with 3-(2-furoyl)quinoline-2-carboxyaldehyde) are effective for primary amines, in some cases effective for secondary amines, and in no cases effective for quaternary amines (Knapp, 1979; Toyo’oka, 1999). Hence, the absence of quaternary ammonium compounds in previous studies is most probably because they were not derivatised and thus were not detected. The profile of organic N molecules in soil reflected that soil can be an osmotically challenging environment. Samples were collected when soil was moist, but based on water content measured in previous years it is probable the soil was exposed to several dryrewet cycles during the preceding three months of summer (Warren and Taranto, 2011). When soil dries microbes and plants avoid dehydration by accumulating compatible solutes, whereas when dry soil is rewet microbes rapidly release accumulated solutes to avoid a rapid influx of water into cells, drastic increase in turgor and possible cellular rupture (Schimel et al., 2007). Not surprisingly many of the most abundant metabolites were compatible solutes (osmolytes) which can accumulate to high concentrations and not affect protein structure or disrupt enzyme activity, for example, proline, quaternary ammonium compounds (carnitine, acetyl carnitine, betaine, choline, ergothioneine), ectoine, and trimethylamine N oxide (Tanret, 1909; Audley and Tan, 1968; Lippert and Galinski, 1992; Hasegawa et al., 2000; Wood et al., 2001). In general, microbes can accumulate compatible solutes either by de novo synthesis or by uptake from the extracellular medium (e.g. soil
solution) (Wood et al., 2001). The large amounts of compatible solutes in the soil solution indicate that uptake from the soil solution could be an important pathway for microbial accumulation of compatible solutes in addition to a possible source of N for uptake by higher plants. The second largest pool of compounds was 42 molecules that could not be identified (Table S1), and unknowns were six of the 25 most abundant non-peptides. On the one hand this highlights how much remains to be learnt, but on the other hand it is not surprising: metabolomic analyses of single species commonly report more than 50% of metabolites are unknowns (Lisec et al., 2006; Sugimoto et al., 2010). Teenty-four of the unknowns have nominal masses matching those of di-peptides, and thus a maximum of 24 unknowns could be di-peptides and a minimum of 18 unknowns are non-peptides. Until such time as the unknowns can be identified, or at the very least placed in broad classes, there will be large uncertainty in the total size of non-peptide versus peptide pools. The aim of this study was not to identify peptides because the method is sub-optimal for peptides; nevertheless, 14 peptides were at least partially identified based on MS/MS spectra. This is clearly an underestimate of di-peptide diversity because at least some of the unknowns are probably di-peptides and, more importantly, the scan range of m/z 50e250 meant that it was possible to measure fewer than 50% of the possible di-peptide combinations. Direct measurement of molecular forms of peptides are timely because recent studies have suggested that the pool of small peptides (less than 1 kDa) may be as large or larger than the pool of amino acids (e.g. Farrell et al., 2011a, b). To date we know almost nothing about the molecular forms of peptides in soil water, but this information is important given that the behavior of peptides will vary depending upon number and nature of amino acid residues. For amino acids we know that acidic, neutral and basic amino acids diffuse through the soil at different rates (Owen and Jones, 2001) and by analogy the rate of diffusion of peptides will be a function of the number of acidic, neutral and basic amino acids. An extra layer of complexity with peptides is that solubility varies with the number and nature of amino acid residues such that even short peptides of five amino acids can be insoluble if all amino acids are hydrophobic. Clearly, many of the questions regarding the importance of peptides to N cycling and plant N nutrition require knowledge of the molecular nature of peptides. 4.3. Limitations Additional studies on other soils are required to establish whether the profile of small organic N molecules reported here is reflected in other soils. In this context it is worth noting that the identified molecules are products of ubiquitous microbial physiology and biochemistry (Lippert and Galinski, 1992; Hasegawa et al., 2000; Wood et al., 2001) and/or have previously been described from soil (e.g. ergothioneine, Tanret, 1909; Audley and Tan, 1968). The measurements reported here almost certainly underestimate the number of small organic N molecules for four reasons. First, measurements were made on soil at one time of the year under one set of conditions, but it is probable that the total number of molecules would increase if samples were also collected at other times of the year or under abiotic stresses. For example, during summer drought, winter frost or waterlogging one would expect large changes in profiles of organic N in the short-term due to upregulation of some metabolic pathways and downregulation of others (e.g. associated with osmoregulation, cryotolerance, oxidative vs. reductive metabolism) and in the longer-term due to changes in composition of the microbial community. Second, the analytical approach cannot analyse organic N molecules that are neutral or anionic at low pH, e.g. N-acetylglucosamine and
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nucleoside mono- di- and tri-phosphates. Third, signals from abundant molecules (e.g. glutamine, glutamate, carnitine, acetyl carnitine) may obscure weaker signals from molecules present at several orders of magnitude smaller concentrations. The final reason that organic N in soil water is more diverse than reported here is because the CE-MS method used here cannot generally separate D- and L-isomers, but one would expect soil water to contain a mixture of D- and L-isomers of amino acids and peptides. 4.4. Implications and recommendations for studies of small organic N Studies of organic N uptake and cycling generally involve addition of isotope labeled organic N molecules (Chapin et al., 1993; Jones and Darrah, 1993; Warren, 2006; Hill et al., 2011 ), and this ought to be guided by knowledge of which forms of organic N occur in soil water. For example, one logical approach would be to study the uptake and cycling of those small organic N molecules that are most abundant in soil water. Previous studies have focused on uptake and cycling of protein amino acids (Chapin et al., 1993; Jones and Darrah, 1993; Warren, 2006) presumably because protein amino acids were assumed to be the most abundant small organic N molecules. However, results of this study indicate that the pool of small organic N in soil water is more diverse and there can be large amounts of several quaternary ammonium compounds and other N-containing compatible solutes. In principle, N-containing compatible solutes could make a significant contribution to ecosystem N cycling based on large concentrations in soil water and the presence in microbes of multiple osmoregulatory transporters with overlapping substrate specificities (Wood et al., 2001), though this needs to be evaluated in future studies. 4.5. Conclusions We currently have an incomplete view of the pool of small organic N in soil water. Until now common amino acids and small peptides have assumed centre stage, but at least for the soil examined in this study the time has come for common amino acids and peptides to step aside and share centre stage with a cast of other organic N molecules such as quaternary ammonium compounds. The profile of organic N molecules in soil reflected that soil can be an osmotically challenging environment. Some of the most abundant molecules were quaternary ammonium compounds and other Ncontaining compatible solutes. In principle, N-containing compatible solutes of microbial and/or plant origin could make a significant contribution to ecosystem N cycling. The exciting next steps are to contrast the profiles of small organic N molecules in soils under a range of biotic and abiotic conditions; and determine the roles of quaternary ammonium compounds and uncommon amino acids in N cycling. Acknowledgements Charles Warren is supported by a Future Fellowship from the Australian Research Council. Dr. Matthias Pelzing is thanked for assisting with set-up of CE-MS. Dr. Maria Taranto collected the soil used in this experiment. The comments of two reviewers helped to greatly improve this manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2012.09.025.
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References Abuarghub, S.M., Read, D.J., 1988. The biology of mycorrhiza in the Ericaceae .12. Quantitative-analysis of individual free amino-acids in relation to time and depth in the soil-profile. New Phytologist 108, 433e441. Andersson, P., Berggren, D., 2005. Amino acids, total organic and inorganic nitrogen in forest floor soil solution at low and high nitrogen input. Water, Air and Soil Pollution 162, 369e384. Audley, B.G., Tan, C.H., 1968. The uptake of ergothioneine from the soil into the latex of Hevea brasiliensis. Phytochemistry 7, 1999e2000. Baidoo, E.E.K., et al., 2008. Capillary electrophoresis-Fourier transform ion cyclotron resonance mass spectrometry for the identification of cationic metabolites via a pH-mediated stacking-transient isotachophoretic method. Analytical Chemistry 80, 3112e3122. Baran, R., et al., 2006. MathDAMP: a package for differential analysis of metabolite profiles. BMC Bioinformatics 7. Bino, R.J., et al., 2004. Potential of metabolomics as a functional genomics tool. Trends in Plant Science 9, 418e425. Chalcraft, K.R., Britz-McKibbin, P., 2009. Newborn screening of inborn errors of metabolism by capillary electrophoresis-electrospray ionization-mass spectrometry: a second-tier method with improved specificity and sensitivity. Analytical Chemistry 81, 307e314. Chapin, F.S., Moilanen, L., Kielland, K., 1993. Preferential use of organic nitrogen for growth by a nonmycorrhizal Arctic sedge. Nature 361, 150e153. Farrell, M., Hill, P.W., Farrar, J., Bardgett, R.D., Jones, D.L., 2011a. Seasonal variation in soluble soil carbon and nitrogen across a grassland productivity gradient. Soil Biology & Biochemistry 43, 835e844. Farrell, M., Hill, P.W., Wanniarachchi, S.D., Farrar, J., Bardgett, R.D., Jones, D.L., 2011b. Rapid peptide metabolism: A major component of soil nitrogen cycling? Global Biogeochemical Cycles 25. Harada, K., Fukusaki, E., 2009. Profiling of primary metabolite by means of capillary electrophoresis-mass spectrometry and its application for plant science. Plant Biotechnology 26, 47e52. Hasan, M.N., Park, S.H., Oh, E., Song, E.J., Ban, E., Yoo, Y.S., 2010. Sensitivity enhancement of CE and CE-MS for the analysis of peptides by a dynamic pH junction. Journal of Separation Science 33, 3701e3709. Hasegawa, P.M., Bressan, R.A., Zhu, J.K., Bohnert, H.J., 2000. Plant cellular and molecular responses to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology 51, 463e499. He, H.B., Xie, H.T., Zhang, X.D., 2006. A novel GCMS technique to assess N-15 and C13 incorporation into soil amino sugars. Soil Biology & Biochemistry 38, 1083e 1091. Hill, P.W., et al., 2011. Acquisition and assimilation of nitrogen as peptide-bound and D-enantiomers of amino acids by wheat. Plos One 6. Horai, H., et al., 2010. MassBank: a public repository for sharing mass spectral data for life sciences. Journal of Mass Spectrometry 45, 703e714. Inselsbacher, E., Ohlund, J., Jämtgård, S., Huss-Danell, K., Näsholm, T., 2011. The potential of microdialysis to monitor organic and inorganic nitrogen compounds in soil. Soil Biology & Biochemistry 43, 1321e1332. Ivarson, K.C., Sowden, F.J., 1969. Free amino acid composition of the plant root environment under field conditions. Canadian Journal of Soil Science 49, 121e 127. Jämtgård, S., Näsholm, T., Huss-Danell, K., 2010. Nitrogen compounds in soil solutions of agricultural land. Soil Biology & Biochemistry 42, 2325e2330. Jones, D.L., Darrah, P.R., 1993. Influx and efflux of amino-acids from Zea mays L roots and their implications for N-nutrition and the rhizosphere. Plant and Soil 156, 87e90. Kaspar, H., et al., 2009. Urinary amino acid analysis: A comparison of iTRAQ (R)-LCMS/MS, GC-MS, and amino acid analyzer. Journal of Chromatography B e Analytical Technologies in the Biomedical and Life Sciences 877, 1838e1846. Kielland, K., 1995. Landscape patterns of free amino acids in arctic tundra soils. Biogeochemistry 31, 85e98. Knapp, D.R., 1979. Handbook of Analytical Derivatization Reactions. John Wiley and Sons, New York. Lippert, K., Galinski, E.A., 1992. Enzyme stabilization by ectoine-type compatible solutes e protection against heating, freezing and drying. Applied Microbiology and Biotechnology 37, 61e65. Lisec, J., Schauer, N., Kopka, J., Willmitzer, L., Fernie, A.R., 2006. Gas chromatography mass spectrometry-based metabolite profiling in plants. Nature Protocols 1, 387e396. Oikawa, A., Fujita, N., Horie, R., Saito, K., Tawaraya, K., 2011. Solid-phase extraction for metabolomic analysis of high-salinity samples by capillary electrophoresismass spectrometry. Journal of Separation Science 34, 1063e1068. Owen, A.G., Jones, D.L., 2001. Competition for amino acids between wheat roots and rhizosphere microorganisms and the role of amino acids in plant N acquisition. Soil Biology & Biochemistry 33, 651e657. Patti, G.J., Yanes, O., Siuzdak, G., 2012. Metabolomics: the apogee of the omics trilogy. Nature Reviews Molecular Cell Biology 13, 263e269. Read, D.J., Bajwa, R., 1985. Some nutritional aspects of the biology of ericaceous mycorrhizas. Proceedings of the Royal Society of Edinburgh 85B, 317e332. Roberts, P., Jones, D.L., 2008. Critical evaluation of methods for determining total protein in soil solution. Soil Biology & Biochemistry 40, 1485e1495. Schimel, J., Balser, T.C., Wallenstein, M., 2007. Microbial stress-response physiology and its implications for ecosystem function. Ecology 88, 1386e1394.
450
C.R. Warren / Soil Biology & Biochemistry 57 (2013) 444e450
Schulten, H.R., Schnitzer, M., 1997. The chemistry of soil organic nitrogen: a review. Biology and Fertility of Soils 26, 1e15. Smith, C.A., et al., 2005. METLIN e a metabolite mass spectral database. Therapeutic Drug Monitoring 27, 747e751. Soga, T., Heiger, D.N., 2000. Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry. Analytical Chemistry 72, 1236e1241. Soga, T., Ohashi, Y., Ueno, Y., Naraoka, H., Tomita, M., Nishioka, T., 2003. Quantitative metabolome analysis using capillary electrophoresis mass spectrometry. Journal of Proteome Research 2, 488e494. Soper, F.M., Paungfoo-Lonhienne, C., Brackin, R., Rentsch, D., Schmidt, S., Robinson, N., 2011. Arabidopsis and Lobelia anceps access small peptides as a nitrogen source for growth. Functional Plant Biology 38, 788e796. Streeter, T.C., Bol, R., Bardgett, R.D., 2000. Amino acids as a nitrogen source in temperate upland grasslands: the use of dual labelled (C-13, N-15) glycine to test for direct uptake by dominant grasses. Rapid Communications in Mass Spectrometry 14, 1351e1355. Sugimoto, M., Hirayama, A., Robert, M., Abe, S., Soga, T., Tomita, M., 2010. Prediction of metabolite identity from accurate mass, migration time prediction and isotopic pattern information in CE-TOFMS data. Electrophoresis 31, 2311e2318. Tanret, C., 1909. The new base drawn from rye ergot, ergothioneine. Comptes Rendus Hebdomadaires Des Seances De L Academie Des Sciences 149, 222e224.
Toyo’oka, T., 1999. Modern Derivatization Methods for Separation Sciences, England. Turnbull, M.H., Schmidt, S., Erskine, P.D., Richards, S., Stewart, G.R., 1996. Root adaptation and nitrogen source acquisition in natural ecosystems. Tree Physiology 16, 941e948. Warren, C.R., 2006. Potential organic and inorganic N uptake by six Eucalyptus species. Functional Plant Biology 33, 653e660. Warren, C.R., 2008. Rapid and sensitive quantification of amino acids in soil extracts by capillary electrophoresis with laser-induced fluorescence. Soil Biology & Biochemistry 40, 916e923. Warren, C.R., Taranto, M.T., 2010. Temporal variation in pools of amino acids, inorganic and microbial N in a temperate grassland soil. Soil Biology & Biochemistry 42, 353e359. Warren, C.R., Taranto, M.T., 2011. Ecosystem respiration in a seasonally snowcovered subalpine grassland. Arctic Antarctic and Alpine Research 43, 137e146. Werdin-Pfisterer, N.R., Kielland, K., Boone, R.D., 2009. Soil amino acid composition across a boreal forest successional sequence. Soil Biology & Biochemistry 41, 1210e1220. Wishart, D.S., et al., 2007. HMDB: the human metabolome database. Nucleic Acids Research 35, D521eD526. Wood, J.M., et al., 2001. Osmosensing and osmoregulatory compatible solute accumulation by bacteria. Comparative Biochemistry and Physiology a-Molecular and Integrative Physiology 130, 437e460.