A proteomics approach to study in vivo protein Nα-modifications

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A proteomics approach to study in vivo protein N α -modifications Xumin Zhang, Juanying Ye, Peter Højrup⁎ Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark

AR TIC LE I N FO

ABS TR ACT

Article history:

In this article we present a simple method to enrich peptides containing in vivo Nα-modified

Received 15 July 2009

protein N-termini. We demonstrate that CNBr-activated Sepharose, a commercial amine

Accepted 9 September 2009

reactive matrix, can selectively couple peptides via the α-NH2 group under mild conditions. Following digestion by trypsin, a simple incubation step with the CNBr-activated Sepharose

Keywords:

by which the free α-NH2 containing peptides are coupled with matrix through a covalent

Protein Nα-modifications

bond, allows the separation of Nα-modified peptides from massive free α-NH2 containing

Acetylation

peptides. The removal of contaminant peptides with artificial Nα-modifications, like

Formylation

cyclization of N-terminal S-carbamoylmethylcysteine and glutamine, are also discussed.

Propionylation

Application of this method to tryptic digests of HeLa cell proteins resulted by a single LC-MS/

CNBr-activated Sepharose 4B

MS analysis in the identification of 588 in vivo Nα-modified peptides, of which 507 contain IPI

Lys-N

(International Protein Index) annotated protein N-termini and 81 contain IPI unannotated protein N-termini. Most of the identified modifications are acetylations with only a few formylations and propionylations present. Furthermore, Lys-N digestion was also applied and resulted in the identification of 394 in vivo Nα-modified peptides, of which 371 contain IPI annotated protein N-termini and 23 contain IPI unannotated protein N-termini. Combination of the two datasets leads to the identification of 675 Nα-modified IPI annotated protein N-termini and 88 Nα-modified IPI unannotated protein N-termini. Our results suggest that N-terminal acetyltransferases (NATs) may function as N-terminal formyltransferases (NFTs) and N-terminal propionyltransferases (NPTs) in vivo. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

Bottom-up proteomics has been widely used for high throughput protein identification. In this approach, the protein is cleaved into characteristic fragments by specific proteases, usually trypsin, and then analyzed by mass spectrometry (MS) or tandem mass spectrometry (MS/MS). The overwhelming sample complexity, due to numerous proteins, large number of modifications and vast diversity in their abundances (both

proteins and modifications), is always a great challenge in practical proteomic studies. Since it is widely accepted that a protein may be characterized based on single peptide with well matched mass spectrum [1,2], enrichment and identification of only one characteristic peptide from each protein can greatly reduce the sample complexity. To date more than 200 posttranslational modifications (PTM) have been discovered [1,3,4]. In eukaryotes, the modifications on α-NH2 of proteins are among the most common, occurring both co- and post-

Abbreviations: ACN, acetonitrile; CHCA, α-cyano-4-hydroxycinnamic acid; ESI, electrospray ionization; LC, liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NATs, N-terminal acetyltransferases; NFTs, N-terminal formyltransferases; NPTs, N-terminal propionyltransferases; SCX, strong cation exchange chromatography. ⁎ Corresponding author. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark. Tel.: +45 6550 2371; fax: +45 6593 1770. E-mail address: [email protected] (P. Højrup). 1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.09.007

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translationally [5]. It is estimated that around 90% of α-NH2 of soluble proteins are blocked in eukaryotic cells, and 80% of this is contributed by Nα-Acetylation [6–8]. Consequently, enrichment and identification of Nα-modified peptides solely can be a promising strategy to facilitate the identification of highly complex protein mixtures, which is still a challenge in global proteomic studies. The biological significance of Nα-acetylation is still being debated. Although a yeast mutant lacking N-terminal acetyltransferases (NATs) is viable, slower growth, partial mating defection, and halted budding when limited for nutrition were observed [9,10]. The result suggests that Nα-acetylation is important although not vital for the organism. Nα-acetylation functions differently for different proteins. It is found to be related to protein assembly, stability, function and location in some studies [11–19]. Therefore a detailed study of protein Nαmodification will increase our knowledge of the biological properties of given proteins. Recently, a series of methods targeting protein N-termini containing peptides have been introduced. Gevaert et al. developed the combined fractional diagonal chromatography (COFRADIC) with assistance of a two step chemical derivatization reaction [20]. First, the free amino groups of proteins (α-NH2 and ε-NH2) are chemically acetylated, and, second, after digestion with trypsin, the digest products are fractionated by HPLC. Each fraction is then submitted to trinitrobenzenesulfonic acid treatment to derivatize nascent α-NH2 with a highly hydrophobic group (the peptides containing protein N-termini are immune to this reaction due to protection by acetylation). Finally, the derivatized products are separated by HPLC. The sub-fractions eluting at the same retention time as before are mainly consisting of the protein N-termini-containing peptides. Besides the some side reactions, i.e. partially acetylation on serine and threonine [20,21], the obvious shortages are the labor and time consumptions from the two step LC separations (many fractions are needed for good purification) and the extensive chemical reactions for each fraction. McDonald et al. simplified this method by using biotin-NHS ester instead of trinitrobenzenesulfonic acid to block the nascent α-NH2 of the tryptic peptides [22]. After incubation with a streptavidin resin, the biotinylated peptides are bound to the resin and thus the protein N-terminus containing peptides can be enriched in the flowthrough fraction. In these two methods, both natively and chemically acetylated peptides are enriched, and it is suggested that the native Nα-acetylated peptides can be discriminated if stable-isotope labeled acetylating reagent is used [22]. Meanwhile, several other chemical derivatizationbased methods have been developed with focus on de novo sequencing of protein N-terminus using pure protein sample [23–26]. These methods were recently reviewed by Meinnel and Giglione [5]. An alternative method for in vivo Nα-modified peptide enrichment is based on strong cation exchange chromatography (SCX) [27,28]. Due to reduced basicity, the Nα-modified tryptic peptides are eluted earlier than other regular tryptic peptides in SCX separation. However, both the efficiency and specificity of this method are questionable as the basicity of peptides does not rely solely on the α-NH2. E.g. Nα-modified peptides with internal basic amino acid, such as histidines and missed cleavage sites, will not be included in the first

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fractions [29], and the peptides with negative-charged modification, such as phosphorylation, can be co-eluted in the first fractions with Nα-modified peptides. It is evident that an amine reactive matrix should be useful in the enrichment as the only significant difference of Nαmodified peptides from the regular peptides is the lack of a free α-amine. The ε-amine from Lys residue is the obstacle due to its chemical similarity to α-amine, thus the enrichment effect can only be achieved, if the reaction with the matrix can distinguish between α-amine and ε-amine. Mikami developed a method for selective isolation of N-blocked peptides by a coupling reaction with homemade isocyanate-resin in acidic condition [30]. However, the method was not applied to any highly complex sample and it was not seen to be used by other researchers, probably because the resin is not commercially available. CNBr-activated Sepharose 4B, a commercial amine reactive matrix, is widely used for coupling bio-molecules via primary amines, and it has been used to enrich N-terminal peptide from an Nα-blocked protein after the blockage of ε-amine group by succinylation [31]. Here we present a simple method for enrichment of in vivo Nα-modified peptides. We demonstrate that under specific conditions, CNBr-activated Sepharose will selectively react with α-NH2 containing peptides and thus the chemical blockage of ε-amine group by succinylation is not necessary. In this way, the α-NH2 containing peptides can be almost quantitatively removed by covalent binding to the matrix, and the Nα-modified peptides are enriched in the flowthrough fraction regardless whether they contain ε-NH2 or not. Application of this method to trypsin and Lys-N digests of HeLa cell extracts resulted in the characterization of 675 Nα-modified IPI annotated protein N-termini and 88 Nα-modified IPI unannotated protein N-termini.

2.

Experimental procedures

2.1.

Materials

Pure water was obtained from a PURELAB Ultra system (ELGA, UK). NaH2PO4, Na2HPO4, NaHCO3, α-cyano-4-hydroxycinnamic acid (CHCA) and bovine serum albumin (BSA) were from Sigma-Aldrich (St. Louis, MO, USA). Modified trypsin was from Promega (Madison, WI). Qcyclase enzyme (50 U/ml) and pGAPase solution (25 U/mL) in TAGZyme™ kit were from Qiagen (Hilden, Germany). Peptidyl-Lys metalloendopeptidase was purchased from Seikagaku Biobusiness Corporation through Associates of Cape Cod. Poros Oligo R3 reversedphase material was from PerSeptive Biosystems (Framingham, MA). GELoader tips were from Eppendorf (Hamburg, Germany). CNBr-activated Sepharose 4B was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and solvents were of the highest commercial quality and were used without further purification. The Protein Center software was from Proxeon Bioinformatics (Odense, Denmark).

2.2.

Protein extraction and digestion of HeLa cell

The clean in-solution digestion was adopted in this work with minor modifications [32]. After washing twice with PBS, the cell pellet was resuspended in 200 μl solution containing 1% sodium

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dodecyl sulfate (SDS) and 5 mM dithiothretol (DTT). The solution was further sonicated for 3 min (2 s sonication time with 5 s intervals) followed by centrifugation at 20,000g for 20 min at 20 °C. The supernatant was collected and submitted to acetone precipitation. Briefly, the supernatant was mixed with 8 volumes of acetone (ice-cold) and kept at −20 °C for 2 h to allow protein to precipitate; the acetone was removed after centrifugation at 20,000g for 20 min at 4 °C. After washing twice with ice-cold acetone, the pellet was completely dried in vacuum and weighed. Since the digestion buffer used in this study lacks detergent and chaotrope, and the resulted slurry solution is not suitable for protein determination, we measured the protein content using a chaotrope containing buffer instead. A small portion of the pellet was weighed and dissolved in chaotrope containing solution (7 M urea + 2 M thiourea) and used for protein determination by Bradford assay. The major portion of the pellet was weighed and resuspended in digestion buffer (200 mM phosphate buffer+ 5 mM DTT, pH 8.0) and further sonicated for 3 min (2 s sonication time with 5 s intervals). The slurry solution was submitted to reduction by 5 mM DTT at 37 °C for 1 h, followed by alkylation with 15 mM acrylamide for 1 h at room temperature. The excess acrylamide was extinguished with an additional 5 mM DTT. The protein solution was divided into two equal portions and digested overnight with trypsin (1:20) or Lys-N (1:20) at 37 °C on an Eppendorf Thermomixer (900 rpm). In our previous experience with cell or tissue extracts, some unknown acid-insoluble material was extracted as well due to the basic extraction buffer, which interferes with the later desalting step carried out under acidic conditions. Therefore, an acidification procedure was employed prior to further analysis. After digestion, ¼ volume of 1 M HCl was added to adjust the pH to ~1.5, and the resulting acid-insoluble fraction was removed by centrifugation at 20,000g for 20 min. ¼ volume of 1 M NaOH was added to adjust the pH back to 7.5–8.0.

2.3.

was added to the resin to elute the non-specific binding peptides, and after 3 min of gentle vortexing, the supernatant was collected as described above. The two supernatant fractions were pooled and the ACN was removed in vacuum. Before further treatment, 2% of the sample was analyzed by MALDI-MS, and the acquired spectrum was compared to that of the original sample. The enrichment procedure should be repeated with half a volume (50 μl) of freshly activated resin for better selectivity, if the intensive peaks in original spectrum can still be observed. It should be noted that the use of excess resin would lead to the loss of Lys-containing peptides.

2.5.

The resulted peptide solution was loaded onto a homemade Poros R3 microcolumn [33]. After washing with 10 μl 1% formic acid, the bound peptides were eluted with 10 μl 30% acetonitrile (ACN) / 0.1% formic acid and then with 10 μl 60% acetonitrile (ACN) / 0.1% formic acid. The two eluates were pooled and dried in vacuum. Prior to LC-MS/MS analysis, the dried peptides were dissolved in 5.5 μl 1% formic acid.

2.6.

MALDI-TOF MS and MS/MS analysis

MALDI-TOF-MS was used for initial screening of the sample quality and composition. Prior to MALDI analysis, the peptide samples were desalted on Poros R3 microcolumns [33], and eluted directly onto the sample supports with matrix solution (5 mg/ml CHCA in 70% ACN, 0.1% TFA). MALDI-MS and MS/MS were performed using a Bruker Ultraflex Tof/Tof MS (Bruker, Bremen, Germany) or a 4800 Proteomics Analyser (Applied Biosystems, Foster City, CA). All spectra were obtained in positive reflector mode and mass spectrometric data analysis was performed using the Bruker Daltonics FlexAnalysis Software (version 2.4) or Data Explorer (version 4.5).

Enzymatic removal of N-terminal pyroglutamate 2.7.

The N-terminal pyroglutamate removal step was carried out just after the overnight tryptic digestion procedure. Before the reaction, pGAPase solution (25 U/mL) was activated by incubation with the same volume of activation buffer (50 mM phosphate buffer + 10 mM DTT, pH 8.0) at 37 °C for 10 min. For 100 μg peptides, 100 mU of pGAPase and 300 mU Qcyclase were used. After the pH was adjusted to ~8.0, the reaction was accomplished by a 1-h incubation with 5 mM DTT at 37 °C and the remaining DTT was extinguished by an additional 10 mM acrylamide.

2.4.

Desalting

Purification of in vivo Nα-modified peptides

The CNBr-activated Sepharose was activated by rehydration and three-time wash in 10 volumes of 1 mM HCl (ice-cold) just before use. A certain volume of activated resin (100 μl bed volume when 100 μg digested protein was applied) was transferred to a new tube, and the supernatant was carefully removed with a GELoader tip. All the above procedures were carried out on ice. An aliquot of digest solution containing 100 μg peptides was added and incubated with the activated resin by a constant, slow end-over-end rotation at room temperature. After 2 h incubation, the supernatant was collected by filtration through a GELoader tip. Additionally, 100 μl acetonitrile (ACN)

Nanoflow LC-ESI-MS/MS

LC-ESI-MS/MS analysis was performed using a nanoflow EASY nLC system (Proxeon A/S, Odense, Denmark) coupled to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific). A one-column system was used in EASY nLC to avoid sample loss during loading step. Samples were loaded and analyzed onto a homemade ~18 cm fused silica column (100 μm i.d.; 375 μm o.d.; Reprosil C18-AQ, 3 μm (Dr. Maisch, AmmerbuchEntringen, Germany)). The mobile phases consisted of Solution A (0.1% formic acid) and Solution B (90% ACN; 0.1% formic acid). After sample loading, sequential elution of peptides was accomplished using a two-step linear gradient of 0% to 34% B in 100 min, 34% to 100% B in 5 min and 100% B for 8 min at a flow rate of 250 nl/min. Data-dependent analysis was employed in MS analysis: the 5 most abundant ions in each MS scan were automatically selected and fragmented, the threshold for fragmentation was set to 105 and dynamic exclusion was set at 45 s.

2.8.

Database search and manual validation

So far, several Nα-modifications in eukaryote have been discovered, including a high content of acetylation and a

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lesser content of formylation, propionylation, myristoylation and palmitoylation [5]. Although N-terminal myristoylation and palmitoylation clearly have significant effects on protein– protein interaction, protein–lipid interaction and protein localization, and are involved in different biological processes [34–37], no myristoylation and palmitoylation was revealed in our datasets, most likely due to their low abundance. These two modifications are thus excluded from the possible modifications for the Mascot search. The raw data were converted into Mascot Generic Files (mgf) using Proteome Discovery (Version 1.0, Thermo Fisher Scientific). The resulting mgf files were searched against the IPI human protein sequence database (20081209, 77,890 sequences) using an in-house Mascot server (Version 2.2.04, Matrix Science, London, UK) with the following parameters: the enzyme was selected as trypsin or Lys-N depending on the sample preparation; only one missed cleavage site is allowed; 5 ppm mass tolerances for MS and 0.8 Da for MS/MS fragment ions; propionamidation on cysteine as fixed modification; protein N-acetylation, protein Nformylation, protein N-propionylation, Pyroglutamate on peptide N-terminal and Oxidation on methionine as variable modifications. After removal of redundant results, the identified peptides were considered to be potential candidates, if the p value was ≤ 0.01 and with rank No. 1. For hits scoring lower than 35, further manual verification was carried out based on MS/MS spectra: first, most of the intense peaks should be explained as y or b-ions; second, at least 4 consecutive y or b-ions, or at least 2 series of 3 consecutive y or b-ions were observed. The detailed false-positive rates (FPRs) in different experiments are shown in Table 1. For the study to elucidate the unexpected protein N-termini the enzyme parameter was changed to semi-trypsin or semi-Lys-N; N-acetylation, N-formylation, and N-propionylation on peptide instead of protein were set as variable modifications.

3.

Results and discussions

3.1.

Experiment scheme

The scheme depicting the experimental procedures is shown in Fig. 1. Firstly, after reduction and alkylation the protein extracts are digested overnight by trypsin or Lys-N; secondly, the digests are incubated with CNBr-activated Sepharose 4B, by which the α-NH2 containing peptides are coupled to the beads via covalent bonds, and the enrichment of the Nαmodified peptides is accomplished by a simple filtration step; finally, the enriched Nα-modified peptides in the flow-through fraction is analyzed by mass spectrometry.

3.2.

Alkylation and digestion buffer

Compared to the conventional method [38], there are two modifications in the sample preparation: alkylation with acrylamide and digestion in chaotrope-free buffer. It was observed that the N-terminal S-carbamoylmethylcysteine, the product of conventional alkylation using iodoacetamide, can de-amidate (–17 Da, loss of the α-amine group) to form a stable six-membered ring structure [39,40], by which the peptide with Cys as N-terminus would escape from the coupling reaction and increase the sample complexity. To solve this problem, we tested an alternative alkylating reagent, acrylamide. In our hands, alkylation with acrylamide can avoid the self-cyclization of N-terminal Cys, most likely because the resulting seven-membered ring structure is much less thermodynamically stable. Fig. 2 shows the results from the tryptic BSA digest using different alkylating reagents. It is clear that for the two peptides: CCTESLVNR (m/z 1138.5 for carbamylmethylation or

Table 1 – Summary of the number of identified peptides and protein N-termini in the different experiments. Experiment

IPI database Decoy FPR (%) Mascot score (p ≤ 0.01) Experiment

Trypsin Total peptides

in vivo N -modified peptides

Total peptides

in vivo Nα-modified peptides

966 36 3.73

507 (474) 1 0.2

519 5 0.96

371 (337) 0 0

>27

Unique modified IPI annotated N-termini Total unique modified IPI annotated N-termini Unique modified IPI unannotated N-termini Total unique modified IPI unannotated N-termini

>26

Semi-Trypsin Total peptides

IPI database Decoy FPR (%) Mascot score (p ≤ 0.01)

Lys-N

α

Semi-Lys-N in vivo Nα-modified peptides

772 18 2.33

Total peptides

432 (375) 7 1.6

in vivo Nα-modified peptides

540 3 0.56

342 (255) 1 0.29

>47

>45

474

337 675

80

Values in parenthesis show the number of unique protein N-termini.

23 88

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Fig. 1 – The experimental scheme for the enrichment of in vivo Nα-modified peptides. After digestion with trypsin, an incubation step with an α-amine specific coupling matrix allows the isolation of in vivo Nα-modified peptides from highly complex peptide mixture.

1166.5 for propionylamidation) and CCAADDKEACFAVEGPK (m/z 1927.8 for carbamylmethylation or 1969.8 for propionylamidation), signals corresponding to self-cyclization product (−17 Da) can be found only in the sample treated with iodoacetamide. Another disadvantage of carbamylmethylation is a +57 Da in product mass, which is close to the mass increase caused by the combination of propionylation and deamidation [28]. Therefore, acrylamide was chosen as alkylation reagent in this study. All the peptide sequences described above are confirmed by LC-MS/MS analysis. Urea is often used in protein extracts due to its ability to increase protein solubility, and its side-effect of derivatization of α-amine are well known [41–43]. In our hands, the side-product can be observed by overnight digestion in a buffer containing as little as 1 M urea. Therefore, urea containing buffer was avoided in this study. A clean, chaotrope-free digestion method was adopted in the application of HeLa cell extract [32]. Another small modification is that Na2HPO4 buffer (pH 8.0) instead of NH4HCO3 was used as digestion buffer, because it has sufficient buffering capacity around pH 8 and does not produce any NH3. This means that the digest can be submitted directly to the coupling reaction without any desalting procedure.

3.3.

Coupling reaction condition

The coupling ability of CNBr-activated Sepharose to amine groups is based on a nucleophilic addition reaction, which means that only a deprotonized amine is reactive. It has been shown that the α-amine and not the ε-amine can be deproto-

nized at pH 8.0 due to the difference in basicity between the α-amine and ε-amine group (pKa of the α-amine and the ε-amine are approximately 7.8 and 11 respectively) [44,45]. Therefore α-amine specific coupling may be achieved when an appropriate pH condition is used. We tested the pH effect using a trypsin digest of an SCX fraction of human placenta proteins (a sample from our ongoing project). The coupling reactions were carried in two different buffers: 100 mM phosphate buffer (pH 8.0) and 100 mM NaHCO3 (~pH 9.5). The results are shown in Fig. 3. The spectra of the enriched fractions at pH 8 (middle panel) and pH 9.5 (bottom panel) are completely different from the spectrum of the original sample (top panel), demonstrating that a selective enrichment has been achieved. The three most abundant peaks, m/z 1563.6, m/z 1242.5 and m/z 870.5, in the middle spectrum were analyzed by Maldi-Tof/Tof-MS. As a result, the peaks at m/z 1563.6 and m/z 870.5 are identified as acADQLTEEQIAEFK (Nα-acetylated peptide from calmodulin, P62158) and di-meVATVSLPR (di-methylated autolysis peptide from trypsin, P00761). Although no confident hit can be obtained for the peak at m/z 1242.5, a peak at m/z 147 in MS/ MS spectrum clearly confirms it is a Lys-containing peptide. The signal of the Arg containing peptide (m/z 870.5) is observed with high relative intensity in the bottom spectrum as well, whereas the signals of the Lys-containing peptides (m/z 1563.6 and m/z 1242.5) are observed with far lower relative intensity. The results thus indicate that coupling at high pH condition is likely to cause a significant loss of Lys-containing Nα-modified peptides.

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Fig. 2 – MALDI spectra of tryptic digests of BSA using different alkylating reagents: Iodoacetamide (top panel) and acrylamide (bottom panel). The inserts illustrate the side products resulting from self-cyclization and the 17 Da difference corresponding to loss of NH3.

Another amine reactive matrix, NHS-activated Sepharose 4FF [21], was tested and a similar result was observed at pH 8.0 compared to pH 9.5. However, this matrix was not chosen in this study due to its rather low sample loading capacity compared to CNBr-activated Sepharose 4B. Therefore, the coupling reaction with CNBr-activated Sepharose 4B at pH 8.0 was employed for the following application.

3.4.

Application to HeLa cell extract

We subsequently applied this method to enrich in vivo Nαmodified protein N-termini from HeLa cell. The HeLa cell proteins were extracted by acetone precipitation and digested using the chaotrope-free digestion method. After a one-step coupling reaction and filtration, our first test with 50 μg tryptic digest revealed 528 peptides with modified N-termini, of which 264 (50%) of them are contributed by internal tryptic peptides with N-terminal pyroglutamate. Since the N-terminal pyroglutamate are mostly produced in vitro, it is necessary to remove them in order to increase the number of identified peptides having in vivo modified N-termini. We further employed an N-terminal pyroglutamate removal treatment using Qcyclase and pGAPase just prior to the coupling reaction. Qcyclase catalyzes the conversion of Nterminal glutamine into pyroglutamate, whereas pGAPase

catalyzes the hydrolysis of N-terminal pyroglutamate. Treatment with both enzymes leads to the efficient removal of the N-terminal glutamine and pyroglutamate from all peptides [29]. As a result, 557 peptides with modified N-termini were now identified starting from 100 μg tryptic digests, of which only 50 can be attributed to N-terminal pyroglutamate. The occurrence of peptides with N-terminal pyroglutamate can be explained by the presence of proline as the second residue for 47 (94%) of these peptides. This structure seems to inhibit the activity of pGAPase. An additional 409 α-amine containing peptides were identified, and an unusual high content of acidic residues (Asp and Glu) was observed in the peptide sequences, particularly in the N-term part. In total, 28.1% of the residues in these peptides are constituted by Asp and Glu; the percentage at the N-terminus and the three penultimate residues is 32.0%, 48.9%, 39.4% and 37.9%, respectively. Thus, for these peptides the α-amine may react with neighboring carboxyl group to form ion pair and give rise to a low coupling efficiency with CNBr-activated Sepharose 4B. The identification results are listed in Supplementary Table 1A, and some typical annotated spectra are shown in Fig. 4. In total, 507 in vivo Nα-modified peptides (representing 474 modified protein N-termini, due to oxidation and missed cleavage sites) were identified, 253 with Lys and 254 with Arg as the C-terminus. Moreover, among the 254 peptides with Arg

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Fig. 3 – Enrichment of in vivo Nα-modified peptide from a SCX fraction of human placenta at different pH values. Top panel: the original sample; middle panel: the enriched fraction at pH 8.0 (100 mM phosphate buffer); bottom panel: the enriched fraction at pH 9.5 (100 mM NaHCO3). The three peaks discussed are marked with asterisk.

as the C-terminus, 32 contained internal Lys due to missed cleavage sites or the KP motif, which is resistant to digestion by trypsin. This result clearly showed that the method was efficient for enrichment of Nα-modified peptides regardless whether they contain ε-NH2 or not. It should be mentioned that the ratio of the sample to matrix is vital for the successful employment of the present method. Use of excess or insufficient matrix resulted in lower percentage of the Lys-containing peptides or higher percentage of α-amine containing peptides. On one hand, the ratio of sample to matrix should be adjusted when a different proteinase is applied since the number of cleaved peptides will be different; i.e. less matrix is needed for Lys-C digests because it produces almost half number of peptides than Trypsin. On the other hand, the protein content varies when different protein determination methods and different digestion methods are employed. Therefore, a test of the ratio of sample to matrix prior to biological sample analysis is central in order to achieve the optimal result. Recently, a peptidyl-Lys metalloendopeptidase (Lys-N) cleaving the amide bond of lysine residues was introduced in proteomic studies for various purposes [26,46–48]. The enzyme caught our interest as the digestion products would be ideal

starting material for the current method. After Lys-N digestion, the peptides with in vivo modified N-termini would not contain any primary amine (α-amine or ε-amine), whereas internal peptides would possess Lys as N-termini thus contain both α-amine and ε-amine. Furthermore, N-terminal cyclization is not likely, as Lys is not a substrate for the cyclization reaction. Performing the reaction using 100 μg Lys-N digest resulted in the identification of 371 in vivo Nα-modified peptides (representing 340 in vivo modified protein N-termini) and 148 regular peptides (Supplementary Table 1B). It is surprising that fewer in vivo Nα-modified peptides were identified compared with that resulting from trypsin digest, although the selectivity increased from 52.5% (507 / 966, trypsin digest) to 71.5% (371 / 519, Lys-N digest). Based on the present data the result could be explained by two observations. First, the ionization efficiency of the Nα-modified peptide resulted from Lys-N digest is rather low, if it does not contain any internal basic residue as a proton receptor, whereas the Nα-modified peptides from the trypsin digest contain at least one basic residue (Lys or Arg) at the Cterminus. In our data many intense singly charged peptide signals in the MS scan were frequently observed throughout the LC-MS/MS analysis, and their sequence information was missing due to the instrument being set to exclude singly

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Fig. 4 – The annotated MS/MS spectra of peptides with different in vivo Nα-modification. (A) Propionylated and (B) acetylated N-terminus were identified from Arginyl-tRNA synthetase; (C) formylated and (D) acetylated N-terminus were identified from NEDD4 family-interacting protein 1.

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charged ions for MS/MS experiment. Second, the cleavage specificity of Lys-N is not as high as trypsin. To investigate the cleavage specificity of the Lys-N, we used a Mascot search with the same dataset using semi-Lys-N as the enzyme. Considering only the identified acetylated peptides with IPI annotated protein N-termini, 51.9% (164 / 316) match the cleavage site specifications of Lys-N. This is far lower than the percentage obtained in the same test using trypsin digest, in which 95.4% (334 / 350) match the cleavage site specifications of trypsin. Moreover, 98 peptides with N-terminal pyroglutamate were identified using the semi-Lys-N in this dataset (Supplementary Table 2B). These observations strongly suggest that Lys-N has a wider cleavage activity than indicated, and more investigations are needed before its wide application in proteomic study.

3.5.

Met-Glu, Met-Asp, Met-Asn and Met-Met-Asn, whereas NatC recognize Met-Ile, Met-Leu, Met-Trp and Met-Phe. We classified the Nα-acetylated peptide dataset into two categories, one with acetylation on Met and one with acetylation on other residues due to Met removal. The amino acid occurrence of the three penultimate sites in the peptides with Nα-acetylated Met is shown in Fig. 5A. Clearly, the residues Glu, Asp, Asn and Leu are predominant in the 2nd residue position, which is consistent with the sequences required by NatB and NatC. The residue occurrences at the 3rd and 4th positions do not show any significant bias for any residue. Regarding the peptides with Met removed, Ala, Ser and to a lesser extent Thr, Gly show high occurrences at the starting position (Fig. 5B), which fulfills the substrate criteria by NatA. For the 3 Nαformylated and 2 Nα-propionylated peptides, their sequences also fit the substrate criteria for NATs.

Nα-acetylation of IPI annotated protein N-termini 3.6.

In this study, 474 and 340 modified protein N-termini were determined using trypsin and Lys-N digests respectively. Combination of the two dataset led to identification of 675 modified N-termini, of which 670 protein N-termini were found to be acetylated only, 1 being formylated only, whereas 2 were found in both acetylated and formylated forms, and 2 in both acetylated and propionylated forms (Supplementary Table 1C). Since there was little information about the Nα-formylation and propionylation and only a few peptides with these two modifications were identified in this study, we further performed data analysis based on the 674 peptides with Nαacetylation. In eukaryotic cells, Met is used as the initiating residue for protein synthesis, and the N-terminal Met removal by methionine aminopeptidases (MAPs) and N-terminal acetylation by N-terminal acetyltransferases (NATs) are by far the most common modifications [8,49,50]. The N-terminal acetylation can thus occur on the N-terminal Met or on the second residue when the N-terminal Met is removed. The sequence requirements of the three known NATs have been elucidated by NAT knockout studies in yeast [50]. NatA works only when the N-terminal Met is cleaved off and mainly recognize N-terminal Ser, Ala, Gly and Thr, while the NatB and NatC also act when the original Met remains. NatB recognizes

Nα-acetylation on IPI unannotated protein N-termini

As the protein N-termini in protein database are mostly based on predictive programs and only a few of them are experimentally verified, we employed Mascot searches specifying semi-trypsin or semi-Lys-N in order to identify N-termini in proteins, that are not annotated in the databases, so-called unannotated protein N-termini. There are some methodology developments towards improving the identification rate using semi-trypsin [20,28]; however, all of these are based on reduction of search possibility to lower the threshold value for identification. In this work we use the standard Mascot search and only accepted the peptide hits with high confidence (p < 0.01, corresponding Mascot score is 47 for semiTrypsin and 45 for semi-Lys-N search). A summary of the identification in the different experiments is shown in Table 1. The semi-trypsin search resulted in the identification of 432 peptides with in vivo Nα-modification, 81 of which contain modification on unannotated protein N-termini (Supplementary Table 2A). Among the 81 peptides, 75 peptides are only Nαacetylated, three are formylated, one is propionylated, and one is both acetylated and propionylated. Similarly, the semi-Lys-N search resulted in 342 peptides with in vivo Nα-modifications, of which 23 contain modified unannotated protein N-termini,

Fig. 5 – The N-terminal amino acid distribution of the Nα-acetylated peptides. (A) The Nα-acetylated peptide with N-terminal Met; (B) the Nα-acetylated peptide with N-terminal Met removed.

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having 22 acetylations and 1 propionylation (Supplementary Table 2B). Together we have identified 88 unannotated protein N-termini, 82 in acetylated form, 4 in formylated form, 1 in propionylated form and 1 in both acetylated and propionylated forms (Supplementary Table 2C). The total of 83 peptides with Nα-acetylated unannotated protein N-termini can be classified into three categories: 28 peptides starting after Met, 24 peptides starting with Met and 31 peptides starting at other positions. For the 28 peptides starting after Met, all of them possess Ala, Ser or Thr as N-terminus, which fulfill the substrate criteria for NatA. These acetylated N-termini are likely to be the second residue in proteins with their N-terminal Met removed. Regarding the 24 peptides starting with Met, 20 of them fulfill the substrate criteria for NatB and NatC, and these acetylated N-termini therefore could be considered as the potential initial positions for protein synthesis. Among the other 31 protein N-termini, 12 of these are Ala, Ser or Thr, which are recognized as the substrate by NatA, and the remaining 19 show abundance of Ile, Asp and Glu, which might be produced by other unknown NATs [50] following posttranslational cleavage. Similar observations were obtained on the 5 unannotated protein N-termini with Nα-formylation or Nαpropionylation, of which 4 are Gly, Ser or Thr and 1 is Asp.

3.7.

Nα-formylation and Nα-propionylation

There are only a few reports on the identification of in vivo Nαformylation and Nα-propionylation. Five Nα-propionylated peptides [28] and one Nα-formylated peptide [51] have been revealed by other researcher using MS-based technologies. In this work, we identified six Nα-formylated peptides and four Nα-propionylated peptides in total. Among them only one was identified before. Interestingly, the N-termini of the 15 peptides all show sequence similarity to that of the identified acetylated peptides. Recently, an in vitro study confirmed that two acetyltransferases, p300 and CREB-binding protein could catalyze lysine propionylation in histones [52]. Our result would suggest that the N-terminal acetyltransferases (NATs) may also function as N-terminal formyltransferases (NFTs) and N-terminal propionyltransferases (NPTs) in vivo.

3.8.

Classification of the in vivo N-terminal modified proteins

In total, 720 proteins with in vivo Nα-modifications were identified. We employed ProteinCenter software to analyze

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the molecular function and cellular components of the identified proteins based on the gene ontology (GO) categories and the results are shown in Fig. 6. It should be noted that a protein can be counted multiple times if it possesses multiple functions or is located in several cellular components. As showed in Fig. 6A, a majority of the identified proteins have been assigned the molecular functions of protein binding or catalytic activity. Compared to the results of the identified proteins from an unenriched sample (data not shown), most of the categories show good relative normalized correlations (0.6–1.3) except for the unannotated proteins, where the ratio of the enriched proteins is three fold of that of the untreated sample. Fig. 6B shows the number of proteins in different cellular components. Cytoplasm, nucleus and membrane are the dominant cellular locations. Compared to the results obtained from the untreated sample, most of the category ratios are comparable although with a few exceptions. The ratio of unannotated proteins is more than twice that from the untreated sample, and on the contrary, the ratios of proteins in the endoplasmic reticulum, mitochondrion and ribosome are around 0.4–0.5, while the ratio of the cell surface proteins (only 3 proteins) is about 0.2. Based on the current data we cannot determine whether the variations in the ratios is a result of a specific preference of the current method or presents actual differences in the ratio of modified versus non-modified N-termini. However, it does seem that there is no difference when analyzing for molecular function, while there is a pronounced lower ratio of modified N-termini in certain cellular compartments. The low ratio of the cell surface proteins could be caused by a dominance of N-terminal modifications with a hydrophobic moiety (e.g. myristoylation) that would be difficult to detect with the current method.

4.

Conclusion

A simple method for the enrichment of natively Nα-modified peptides was developed in this work. We have demonstrated that the enrichment of Nα-modified peptides can be achieved by a simple incubation step with CNBr-activated Sepharose, since it can specifically couple α-NH2 containing peptides under controlled conditions. The effectiveness of the procedure is demonstrated by the observation that nearly half of the enriched in vivo N-term modified peptides from the tryptic

Fig. 6 – The annotated information of the identified in vivo Nα-modified proteins. The classification is based on GO molecular functions (A) and cellular components (B). The values indicate the number of proteins which are sorted into each category.

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digest possessed Lys in the C-terminus. Furthermore, the alkylation with acrylamide and pyroglutamate removal treatment can facilitate the enrichment by reducing the artificial Nα-modification caused by cyclization of N-terminal Gln and Cys. Compared to previous methods [20–22,28], the current methods offer a simple operation, only two hours of incubation and the ability to work with very complex samples. However, to get optimal results the ratio of beads to sample needs to be tested beforehand. Application of this method using trypsin and Lys-N digests of HeLa cell extract resulted in the characterization of 675 Nαmodified IPI annotated protein N-termini and furthermore identified 88 Nα-modified IPI unannotated protein N-termini indicating either a relatively high rate of alternative protein initiation or unknown proteolytic activity. Sequence comparison of peptides with different modifications suggests that the NATs may function as NFTs and NPTs on co-translational and post-translational levels.

[11]

[12]

[13]

[14]

[15]

[16]

Acknowledgements [17]

Dr. Clifford Young is acknowledged for the introduction to LCMS/MS analysis. Henrik Larsen and Kasper Engholm-Keller are acknowledged for supplying biological samples. Lene Jakobsen, Søren Andersen and Andrea Lorentzen are acknowledged for their instrument assistance.

[18]

[19]

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jprot.2009.09.007.

[20]

[21]

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