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Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography–digestion–liquid chromatography–electrospray mass spectrometry
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 346 (2005) 150–157 www.elsevier.com/locate/yabio
Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography–digestion–liquid chromatography–electrospray mass spectrometry Jeroen Carol a,b,¤, Maarten C.J.K. Gorseling a, Camiel F. de Jong a, Henk Lingeman a, Charles E. Kientz b, Ben L.M. van Baar b, Hubertus Irth a a
Department of Analytical Chemistry and Applied Spectroscopy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands b TNO Defense, Security, and Safety, P.O. Box 45, 2280 AA Rijswijk, The Netherlands Received 14 June 2005 Available online 7 September 2005
Abstract A multidimensional analytical method for the rapid determination and identiWcation of proteins has been developed. The method is based on the size-exclusion fractionation of protein-containing samples, subsequent on-line trypsin digestion and desalination, and reversed-phase high-performance liquid chromatography–electrospray mass spectrometry detection. The present system reduces digestion times to 20 min and the total analysis time to less than 100 min. Using bovine serum albumin and myoglobin as model proteins, optimization of key parameters such as digestion times and interfacing conditions between the diVerent pretreatment steps was performed. The automated system was tested for the identiWcation of infectious disease agents such as cholera toxin and staphylococcal enterotoxin B. This resulted typically in a positive identiWcation by a total sequence coverage of approximately 40%. 2005 Elsevier Inc. All rights reserved. Keywords: On-line digestion; Biological toxins; Size-exclusion chromatography; Liquid chromatography; Mass spectrometry
The fast identiWcation of infectious disease agents, viruses, bacteria, and protein toxins, is of general importance, for example, in the case of a possible terrorist biological attack [1]. Classical microbiological methods, typically covered in medical microbiology textbooks (for example [2]), frequently take days. Widely accepted serological and immunochemical methods require incubation time and availability of suitable antisera or antibodies. Moreover, these methods are prone to crossreactivity and, therefore, to misidentiWcation (see for example [3], on Brucella and Yersinia). Since the mid 1980s, molecular biological methods, for example based
*
Corresponding author. Fax: +31 20 598 7543. E-mail address: [email protected] (J. Carol).
0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.08.023
on PCR [4], have found wide application. This nucleotide-based identiWcation is highly sensitive and has a selectivity that can be tailored. More recently, proteomics-type mass spectrometry (MS) found application in the Weld of microorganism investigations (see for example [5–7]). The “shotgun” proteomics MS approach [8] addresses protein sequence information and, therefore, attains the speciWcity of PCR-based methods. Although the replication implicit in PCR-based identiWcation methods puts protein identiWcation on the disadvantage with respect to sensitivity, protein structure information from MS is obtained in a generic way. Therefore, we pursue the identiWcation of microorganisms by a shotgun proteomics MS approach. IdentiWcation of bacteria and viruses, to be seen as complex mixtures of proteins, is diYcult in shotgun proteomics
Determination of denaturated proteins and biotoxins / J. Carol et al. / Anal. Biochem. 346 (2005) 150–157
MS. A protein separation step can resolve that diYculty. Presently, two-dimensional (2D)1 polyacrylamide gel electrophoresis (2D-PAGE) is most widely used for the investigation of the proteome of bacteria and viruses. Gelbased separation methods follow well-established protocols that result in a high resolution for proteins unmatched by any other biochemical separation method. However, the preparation of proteins from gels, for subsequent MS identiWcation, is a time-consuming and laborious procedure. Also, 2D-PAGE intrinsically gives problems with regard to reproducibility [9,10]. 2D-liquid chromatography (2D-LC) approaches can well be used to overcome these reproducibility problems [11–13]. This also provides a direct way of MS coupling, with a concomitant reduction in protein and peptide loss. Therefore, 2D-LC MS is potentially better suited than 2D-PAGE and MS, for proteome investigations. Protein sequence information in 2D-LC MS is generally obtained after trypsin digestion and by MS/MS. Trypsin digest peptides generally have a single basic amino acid (trypsin cleavage sites are K and R) that aVords protonation and direction of MS/MS fragmentation. Trypsin digestion methods are commonly performed oV-line from LC-MS(/MS), in solution [14] or in-gel [15,16]. Typical trypsin digestion times for complete digestion range from 4 h to overnight. Shorter digestion times are desired for the rapid identiWcation of infectious organisms, particularly in life-threatening diseases. Here, we report on the development of a multidimensional on-line LC-MS method as a Wrst step towards a general method. Protein separation, prior to digestion LCMS(/MS), is accomplished through on-line coupling with a size-exclusion chromatography (SEC) column. The utility of SEC in proteomics was recently pointed out [17]. The present on-line SEC-digestion-LC-MS system allows injection of protein solutions in denaturing chaotropic buVer. In addition, a digestion step is implemented on-line with an analytical LC peptide separation by means of an immobilized trypsin cartridge and a desalination step on a trapping column. Cartridge trypsin digestion allows faster processing times, typically on the order of 10–60 min. This paper discusses system design issues, typical performance, and a real life application to cholera toxin (CTx) and staphylococcal enterotoxin B (SEB). Experimental Reagents Bovine serum albumin (BSA), 97.0%, was purchased from Fluka (Buchs, Switzerland). Horse heart myoglobin and iodoacetamide were purchased from Sigma (St. Louis, 1 Abbreviations used: 2D, two-dimensional; SEC, size-exclusion chromatography; CTx, cholera toxin; SEB, staphylococcal enterotoxin B; BSA, bovine serum albumin; ESI, electrospray ionization; BMS, bis(2-mercaptoethyl)sulfone; TIC, total ion current; LOD, limit of detection.
151
MO, USA). Cholera toxin and staphylococcal enterotoxin B were obtained from List Biological Laboratories (Campbell, CA, USA). Sequencing-grade modiWed trypsin was obtained from Promega Benelux (Leiden, The Netherlands). Acetonitrile and calcium chloride were obtained from J.T. Baker (Deventer, The Netherlands). Formic acid and ammonium bicarbonate were from Riedel-de-Haën (Seelze, Germany). Bis(2-mercaptoethyl)sulfone was purchased from Toronto Research Chemicals (North York, ON, Canada). Enzymegrade tris(hydroxylmethyl)aminomethane (Tris) was obtained from Ultrapure, Bethesda Research Laboratories (Gaithersburg, MD, USA). Milli-Q water was obtained from a local Milli-Q Academic system (Millipore, Bedford, MA). All solvents were Wltered and degassed before use. Instrumentation A schematic of the chromatographic setup is given in Fig. 1. The LC-ESI MS unit consisted of a micropump (pump 2) (Intelligent Pump 301M; Flom, Tokyo, Japan), a Luna C18(2) column (100 £ 2.1 mm 3 m; Phenomenex, Bester, Amstelveen, The Netherlands), and a Quattro II mass spectrometer (Micromass, Wythenshawe, Manchester, UK). For a determination of limits of detection a Q-TOF mass spectrometer (Micromass) was used. The LC column was mounted in a column oven kept at 45 °C. The SEC unit consisted of a Lab Alliance pump (pump 1) (Instrument Solutions, Nieuwegein, The Netherlands) coupled to a Biosep 2000 SEC column (3.0 £ 4.6 mm; Phenomenex) in a column oven at 45 °C and an LC 2010 C UV detector set at 214 nm (Shimadzu, ’s Hertogenbosch, The Netherlands). For injection, the SEC unit was equipped with an LC 2010 C autoinjector (Shimadzu). The trypsin cartridge was a Poroszymeimmobilized trypsin digestion cartridge (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). The trapping unit consisted of an LC 2010 C pump (pump 3; Shimadzu) and a C18-ODS guard column (10 £ 2.1 mm; Phenomenex). Sample pretreatment Protein denaturation, disulWde reduction, and thiol derivatization were done by a known protocol [14]. BrieXy, a solution containing 200 L Rapigest SF (0.2% v/v in 50 mM NH4HCO3) and 200 L bis(2-mercaptoethyl)sulfone (2 mg/mL) (BMS) was mixed in an Eppendorf vial and 400 L of a protein solution (100 M) was added. This mixture was incubated for 45 min at 58 °C in the dark, after which the mixture was cooled to room temperature. Then 100 L of iodoacetamide (70 mg/mL) was added and the mixture was placed in a water bath of 37 °C, for 0.5 h. The sample mixture was kept for a maximum of 2 weeks in the refrigerator at 4 °C until use. On-line digestion and analytical separation setup Separation of the denatured, derivatized proteins from the denaturation and derivatization reagents was accom-
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Determination of denaturated proteins and biotoxins / J. Carol et al. / Anal. Biochem. 346 (2005) 150–157
Fig. 1. Chromatographic setup. The system consist of two automatedswitching six-port valves, two isocratic pumps (pumps 1 and 2), one gradient pump (pump 3), an auto injector, a size-exclusion column (SEC), a UV detector, a trypsin cartridge, a trapping column, an analytical LC column, and a mass spectrometer.
plished by SEC. Typically, 50 L of the denatured, derivatized protein solution was injected and SEC was performed with the trypsin digest buVer (50 mM Tris and 10 mM CaCl2, adjusted to pH 8.0) as the eluent, at a Xow rate of 50 L/min. The UV detector was set at 214 nm. The sample Xow through the SEC column, trypsin digest cartridge, and analytical LC column was established and controlled with the use of two six-port switching valves (Prospekt 1, Spark Holland, Emmen, The Netherlands), as shown in Fig. 1. All components were coupled by blue (0.25mm ID) and red (0.13-mm ID) stripe-coded poly ether ketone tubing (Bester, Amstelveen, The Netherlands). The total analytical procedure encompasses six steps. First, the six-port valve 1 is switched to temporarily transfer all SEC Xow, in digestion buVer 50 L/min, to waste (position 1). Second, valve 1 is switched to load the trypsin cartridge with a selected protein fraction from the SEC column (position 2). Third, valve 1 is switched back to divert the SEC Xow to waste and to allow trypsin digestion to take place during a predetermined stopped Xow period (position 1). Fourth, after this period, the Xow from pump 3 is switched on to
Xush the trypsin cartridge and to trap the digest peptides on the trapping column with a Xow of 50 L/min of digestion buVer (valve 2 in position 1). During the stopped Xow period, the SEC separation is completed. Fifth, valve 1 is switched to position 2, for a desalination wash of the trapping column with water/acetonitrile (95:5 v/v) containing 0.2 v/v-% formic acid and regeneration of the SEC column and the trypsin cartridge. Sixth, valve 2 is switched to transfer the trapped peptides from the trapping column to the LCMS subsystem (position 2). Thus, the LC-MS analysis run completes the overall analytical procedure. The LC gradient was programmed from 100% solvent A (water/acetonitrile, 95:5 v/v with 0.2 v/v-% formic acid) to 80% solvent B (water/ acetonitrile, 5:95 v/v, with 0.2 v/v-% formic acid) in 60 min, at a Xow rate of 0.1 mL/min. The analytical LC system was directly coupled to the ESI interface of a triple-quadrupole MS instrument (Quattro II; Micromass). The Xow rate of the mobile phase, 0.1 mL/min, was compatible with ESI. The mass spectrometer was set to scan from 200 to 1400 Da, scan time of 3.5 s, at a cone voltage of 30 V, a source temperature of 140 °C, and a capillary voltage of 3.7 kV. The ESI nebulizing gas Xow was set at 17 L/h and the drying gas was set at 375 L/h. When a Q-TOF MS was used the system settings chosen were similar to those of the Quattro II MS. Results and discussion Size-exclusion chromatography Separation of a protein mixture in SEC occurs as a function of the average molecular weight of the proteins. Exclusion volumes and derived exclusion times were obtained by injecting a mixture of known proteins directly on the SEC column and using UV detection (214 nm). A continuous Xow of the digest buVer was pumped as the SEC eluent at a Xow rate of 250 L/min. A typical SEC chromatogram is given in Fig. 2, and derived exclusion times are depicted in Fig. 3. The relative standard deviation of the exclusion times of the diVerent proteins was between 0.45 and 1.38% (n D 6). This constitutes a calibration of the SEC subsystem.
Fig. 2. SEC chromatogram of a mixture of three test proteins; the fraction enclosed by the two lines was typically sent to the digest cartridge.
Determination of denaturated proteins and biotoxins / J. Carol et al. / Anal. Biochem. 346 (2005) 150–157
Trapping column
108 107 Blue dextran 2000
MW (Da)
106 Adolase
105
BSA Ovalbumin Myoglobin
104 103
Diazepam Enkephalin
102 101 R2 = 0.9941 0 1.5 2
153
2.5
3
3.5
4
4.5
exclusion volume (mL)
Fig. 3. Calibration graph of exclusion time versus protein molecular weight.
After digestion, the peptides formed were transferred to the trapping column by Xushing the trypsin cartridge with digestion buVer. Generally, peptides have a relatively high aYnity to the C18 material of the trapping column, as compared to the (buVer) salts. Breakthrough volumes were determined by Xushing the trapping column with the LCESI MS eluent used at the beginning of the analytical gradient. Peptide breakthrough occurred at a volume of 1.50 mL. At a Xow of 100 L/min, a 5-min Xush provided suYcient time for desalination. As the Xush volume was well below the breakthrough volume, desalination did not signiWcantly aVect the trapping of the analyte peptides in the present system. LC-MS
Since, with a constant Xow, the exclusion volume is proportional to exclusion time, a fraction with a certain bandwidth around a given average molecular weight (MW) is selected for transfer to the trypsin digestion column. The SEC chromatogram, Fig. 2, shows that the late eluting proteins have a broader peak than the early eluting proteins. As a consequence, a certain Wxed fraction volume passed from the SEC column to the trypsin cartidge represents a more narrow MW range for the highMW proteins than for the low-MW proteins. BSA and myoglobin were used to further investigate the eVect of this MW bandwidth on SEC heart-cutting. In the present system, the volume of the trypsin cartridge is 88 L. From the SEC calibration it is derived for BSA and myoglobin that 88 L of exclusion volume corresponds to MWs of 10 and 7 kDa, respectively. For the toxins studied, SEB (28 kDa) and CTx (A-chain, 29 kDa), the selected exclusion volume is predicted to represent a bandwidth of 8 kDa.
A linear, binary LC gradient of water and acetonitrile was used for the analytical separation of the peptide mixture. Separation starts with elution of the peptides from the trapping column, switched on-line with the LC column, under conditions of the initial eluent composition. The initial eluent composition requires a relatively high concentration of water (95%, v/v), to allow retention of relatively apolar peptides. Acetonitrile (at a minimum of 5% v/v) was employed in the eluent to avoid the formation of gas bubbles during gradient mixing, whereas formic acid (0.2 v-%) was used to prevent secondary adsorption eVects and to provide protons for ESI. A total ion current (TIC) chromatogram was obtained from the digest products by detection with a triple-quadrupole mass spectrometer. The peptide molecular weights were determined from the ESI mass spectra of an LC-MS run by transformation to [M+H]+ ion masses, in a peptide map. The peptide map was then submitted to a Mascot [18] database search.
On-line digestion
Overall setup
To determine the minimum required digestion time, the amount of undigested myoglobin was monitored as a function of time at 5, 10, 20, 30, and 60 min. The peak area of myoglobin without digestion was taken as 100%. It was observed that 70 § 1.5% of the original myoglobin was digested using a 20-min digestion time. This degree of digestion was suYcient for identiWcation by peptide mapping (Mascot score >72, at p < 0.05). Shorter digestion times did not result in identiWcation of myoglobin. That 20 min digestion was appropriate was conWrmed with digestion of BSA, where 30 min digestion time did not produce signiWcant diVerences from 20 min. For comparison, the oV-line (in solution) digestion to a similar endpoint took typically between 4 and 8 h. With the observations made, the minimum digestion time of 20 min was used throughout further experiments; the rate of digestion was suYcient for identiWcation in all cases.
To test the system performance, 10 L of a BSA solution (1 mg/mL) was injected. The TIC chromatogram of that analysis is shown in Fig. 4. BSA digest peptides are clearly resolved and allow derivation of a peptide map. That map gave a positive Mascot hit for BSA, with a protein score of 176 at 58% sequence coverage. A second performance test concerned injection of a mixture of proteins including BSA. The SEC fraction of 60–70 kDa was selected for digestion, whereas the remainder was Xushed to waste. The TIC chromatogram obtained is shown in Fig. 5. The derived peptide map again gave a signiWcant score of BSA (score 137, 56% coverage). The peptides observed were the same as those seen in the analysis of neat BSA. The reproducibility of the performance of the entire system was determined by examination of Wve trypsin digest peptides of BSA (T14, T33, T57, T58, and T82) in multiple analyses (n D 8) and comparison of the variance in reten-
154
Determination of denaturated proteins and biotoxins / J. Carol et al. / Anal. Biochem. 346 (2005) 150–157 23.83
100
27.06
22.30 21.28
35.81
31.05 31.65 17.62
%
33.60 4.79
9.12 41.25 42.53 46.69
Time 50.00
0 10.00
20.00
30.00
40.00
Fig. 4. Total ion chromatogram obtained from the injection of neat (10 L, 20 M) BSA onto the SEC column; the chromatogram is not two dimensional, as it reXects the digestion and analysis of the BSA fraction from SEC. 22.21
100 21.87 18.73
23.91 24.42
21.19 26.80
30.71
28.59
39.81
37.09
%
40.57 2.41
4.02
35.13 41.85 42.95
47.80 44.48 49.84
0 5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
60.00
65.00
Time 70.00
Fig. 5. Total ion chromatogram from the injection of a mixture of proteins (10 L, 25 M of BSA), where the BSA fraction from SEC was transferred online to the digest cartridge; intact BSA was not visible in the chromatographic range displayed.
tion time and abundance. The retention times displayed a relative standard deviation between 1.3 and 1.7%. The standard deviation of the ion abundances was between 13 and 21%. These relatively high standard deviations of the abundances were probably due to variations in the on-line digestion for BSA but did not aVect the identiWcation of the proteins. To compare performance of the overall methodology with diVerent mass spectrometers, the entire chromatographic system was coupled to a Q-TOF mass spectrometer. Chromatograms obtained with the Q-TOF were well comparable to those obtained with the triple quadrupole instrument, and the LC peak shapes in both chromatograms were similar. With the Q-TOF MS the method LOD was 1.14 M for neat BSA and 2.28 M for BSA in
the mixture. This method LOD was one order of magnitude lower than that obtained with the triple-quadrupole instrument, 12 and 18 M, respectively (§1.3 M, n D 3). Toxins Finally, the entire system was used for target compound analysis in two toxin-containing samples. The Wrst target compound was SEB with an average MW of 28.4 kDa. SEB is produced by various strains of Staphylococcus aureus and is considered a potential biological warfare agent. SEB can straightforwardly be identiWed by oV-line sample treatment and LC-MS [19]. Here, the SEC fraction of 25–35 kDa was selected for on-line digestion and chromatography with
Determination of denaturated proteins and biotoxins / J. Carol et al. / Anal. Biochem. 346 (2005) 150–157
A
155
20.34
100
24.42
%
23.57 21.62 25.02 22.72
14.65 4.665.215.98 7.17 6.74 7.51 8.70 10.82 11.93 9.21 13.46 11.25
29.01 26.55
16.60
25.44
19.58 15.92 17.6218.81
28.67 27.99
57
Time 6.00
B
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
ScanES TIC
25.75
100
28.00
29.20 22.90 36.52 20.77 21.78 19.95
26.76 23.51 23.92
17.82 14.97
29.71
18.83
28.09
41.81 30.12 31.14
% 41.30
33.88 32.25 34.90 37.74
39.27 39.78
43.13 43.95
0
Time 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
Fig. 6. Two total ion chromatograms from diVerent samples containing biological toxins. (A) Sample containing SEB (10 L, 25 M); (B) sample containing CTx (10 L, 25 M).
triple-quadrupole MS detection. The TIC chromatogram obtained is shown in Fig. 6A. The peptide map, summarized in Table 1, yielded a Mascot rank 1 match for SEB with a score of 101 and 40% sequence coverage. This provided ample evidence that the sample contained SEB. The second target compound was CTx with A- and Bchain average molecular weights of 29.3 and 11.6 kDa, respectively. CTx is the main toxic product of toxinogenic
strains of Vibrio cholera and the toxin itself is considered a potential biological warfare agent. A molecule of CTx consists of two non-covalently bound units, A(ctive)-chain and B(inding)-chain, which appear in a one-to-Wve ratio. CTx can straightforwardly be identiWed by oV-line sample treatment and LC-MS [20]. Here, the SEC fraction 25– 35 kDa was selected, so only the A-chain was directed toward the digest cartridge. The TIC chromatogram
Table 1 Observed trypsin digest peptides from the sample containing SEB Fraction
Retention time (min)
m/z value detected [M+H]+
m/z value theoretical [M+H]+
Sequence
T1 T3 T4 T7
14.65 22.55 20.34 16.60
1519.73 1070.49 1586.81 950.46
ESQPDPKPDELHK FTGLMENMK VLYDDNHVSAINVK LGNYDNVR
T22
26.55
1278.66
NLLSFDVQTNK
T22-23 T24-25 T25 T26 T29 T35
22.81 21.70 23.66 25.02 24.42 21.53
1518.24 1070.32 1585.34 950.43 950.68 1277.22 1278.32 1404.78 1435.28 1307.08 658.91 1836.26 966.61
1406.76 1436.77 1308.67 659.38 1837.86 966.54
NLLSFDVQTNKK KVTAQELDYLTR VTAQELDYLTR HYLVK LYEFNNSPYTGYIK IEVYLTTK
156
Determination of denaturated proteins and biotoxins / J. Carol et al. / Anal. Biochem. 346 (2005) 150–157
Table 2 Trypsin digest peptides found after Mascot search of the peptide map from the sample containing CTx from V. cholera strain 569B (NCBI CAA41590, gi|48889) Fraction
Retention time (min)
m/z value detected [M+H]+
m/z value theoretical [M+H]+
Sequence
T5 T6 T7 T9 T15 T17 T20 T25
19.95 36.52 33.88 22.80 25.75 26.76 29.20 21.17
845.23 1001.03 1532.79 1449.68 2594.78 1867.74 1029.42 376.20
845.42 1001.42 1532.72 1449.69 2594.22 1867.86 1029.48 376.16
QSGGLMPR GQSEYFDR GTQMNINLYDHAR HDDGYVSTSISLR YYSNLDIAPAADGYGLAGFPPEHR EEPWIHHAPPGCGNAPR FLDEYQSK DEL
obtained is shown in Fig. 6B, whereas the derived peptide map is summarized in Table 2. The Mascot search resulted in rank 1 identiWcation of CTx, with a score of 101 and 40% sequence coverage. This target analysis readily identiWed the presence of CTx. Conclusion A fully automated on-line analytical system was developed for the identiWcation of a size-exclusion-selected protein fraction from a mixture of proteins. The system couples size-exclusion chromatography on-line to a digest cartridge, analytical peptide separation, and MS or MS/ MS detection. The system was used to identify biological toxins by target compound identiWcation. The total analysis time was reduced to 100 min. This comprises 15 min for SEC separation, 20 min for on-line digestion, 5 min for desalination, and 60 min for analytical separation. The system prevents sample handling losses and eliminates tedious sample preparation steps. As a result, this analysis procedure is much faster than oV-line MS-based identiWcation methods for biological warfare agents, which can take up to 2 days. A drawback of the present system setup is that only a single fraction from a complete SEC run can be transferred for digestion and subsequent LC-MS analysis. The remainder of the protein mixture is transferred to waste. In the analysis of MW-limited targets, for example bacterial 60kDa heat shock proteins, this need not be a problem. For more complicated applications, multiple digestion cartridges may be employed to hold fractions during analysis (see, for example [21]). However, this approach is beyond the scope of our present interest. Two toxins, SEB and CTx, were analyzed with the current system and methodology. These toxins were identiWed by database searching, both with a result of 40% sequence coverage. In the near future, the setup will be miniaturized by the incorporation of micro-LC. Miniaturization will allow application of smaller sample volumes and lower LODs, both favorable in case of microorganism identiWcation. The miniaturized system will be employed as a fast and sensitive method for rapid identiWcation of microorganisms.
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[19] C.E. Kientz, A.G. Hulst, E.R.J. Wils, Determination of Staphylococcal Enterotoxin B by on-line (micro) liquid chromatography-electrospray mass spectrometry, J. Chromatogr. A 757 (1997) 51–64. [20] B.L.M. van Baar, A.G. Hulst, E.R.J. Wils, Characterisation of cholera toxin by liquid chromatography-electrospray mass spectrometry, Toxicon 37 (1999) 85–108. [21] G.J. Opiteck, J.W. Jorgenson, R.J. Anderegg, Two-dimensional SEC/ RPLC coupled to mass spectrometry for the analysis of peptides, Anal. Chem. 69 (1997) 2283–2291.
Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography–digestion–liquid chromatography–electrospray mass spectrometry Jeroen Carol a,b,¤, Maarten C.J.K. Gorseling a, Camiel F. de Jong a, Henk Lingeman a, Charles E. Kientz b, Ben L.M. van Baar b, Hubertus Irth a a
Department of Analytical Chemistry and Applied Spectroscopy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands b TNO Defense, Security, and Safety, P.O. Box 45, 2280 AA Rijswijk, The Netherlands Received 14 June 2005 Available online 7 September 2005
Abstract A multidimensional analytical method for the rapid determination and identiWcation of proteins has been developed. The method is based on the size-exclusion fractionation of protein-containing samples, subsequent on-line trypsin digestion and desalination, and reversed-phase high-performance liquid chromatography–electrospray mass spectrometry detection. The present system reduces digestion times to 20 min and the total analysis time to less than 100 min. Using bovine serum albumin and myoglobin as model proteins, optimization of key parameters such as digestion times and interfacing conditions between the diVerent pretreatment steps was performed. The automated system was tested for the identiWcation of infectious disease agents such as cholera toxin and staphylococcal enterotoxin B. This resulted typically in a positive identiWcation by a total sequence coverage of approximately 40%. 2005 Elsevier Inc. All rights reserved. Keywords: On-line digestion; Biological toxins; Size-exclusion chromatography; Liquid chromatography; Mass spectrometry
The fast identiWcation of infectious disease agents, viruses, bacteria, and protein toxins, is of general importance, for example, in the case of a possible terrorist biological attack [1]. Classical microbiological methods, typically covered in medical microbiology textbooks (for example [2]), frequently take days. Widely accepted serological and immunochemical methods require incubation time and availability of suitable antisera or antibodies. Moreover, these methods are prone to crossreactivity and, therefore, to misidentiWcation (see for example [3], on Brucella and Yersinia). Since the mid 1980s, molecular biological methods, for example based
*
Corresponding author. Fax: +31 20 598 7543. E-mail address: [email protected] (J. Carol).
0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.08.023
on PCR [4], have found wide application. This nucleotide-based identiWcation is highly sensitive and has a selectivity that can be tailored. More recently, proteomics-type mass spectrometry (MS) found application in the Weld of microorganism investigations (see for example [5–7]). The “shotgun” proteomics MS approach [8] addresses protein sequence information and, therefore, attains the speciWcity of PCR-based methods. Although the replication implicit in PCR-based identiWcation methods puts protein identiWcation on the disadvantage with respect to sensitivity, protein structure information from MS is obtained in a generic way. Therefore, we pursue the identiWcation of microorganisms by a shotgun proteomics MS approach. IdentiWcation of bacteria and viruses, to be seen as complex mixtures of proteins, is diYcult in shotgun proteomics
Determination of denaturated proteins and biotoxins / J. Carol et al. / Anal. Biochem. 346 (2005) 150–157
MS. A protein separation step can resolve that diYculty. Presently, two-dimensional (2D)1 polyacrylamide gel electrophoresis (2D-PAGE) is most widely used for the investigation of the proteome of bacteria and viruses. Gelbased separation methods follow well-established protocols that result in a high resolution for proteins unmatched by any other biochemical separation method. However, the preparation of proteins from gels, for subsequent MS identiWcation, is a time-consuming and laborious procedure. Also, 2D-PAGE intrinsically gives problems with regard to reproducibility [9,10]. 2D-liquid chromatography (2D-LC) approaches can well be used to overcome these reproducibility problems [11–13]. This also provides a direct way of MS coupling, with a concomitant reduction in protein and peptide loss. Therefore, 2D-LC MS is potentially better suited than 2D-PAGE and MS, for proteome investigations. Protein sequence information in 2D-LC MS is generally obtained after trypsin digestion and by MS/MS. Trypsin digest peptides generally have a single basic amino acid (trypsin cleavage sites are K and R) that aVords protonation and direction of MS/MS fragmentation. Trypsin digestion methods are commonly performed oV-line from LC-MS(/MS), in solution [14] or in-gel [15,16]. Typical trypsin digestion times for complete digestion range from 4 h to overnight. Shorter digestion times are desired for the rapid identiWcation of infectious organisms, particularly in life-threatening diseases. Here, we report on the development of a multidimensional on-line LC-MS method as a Wrst step towards a general method. Protein separation, prior to digestion LCMS(/MS), is accomplished through on-line coupling with a size-exclusion chromatography (SEC) column. The utility of SEC in proteomics was recently pointed out [17]. The present on-line SEC-digestion-LC-MS system allows injection of protein solutions in denaturing chaotropic buVer. In addition, a digestion step is implemented on-line with an analytical LC peptide separation by means of an immobilized trypsin cartridge and a desalination step on a trapping column. Cartridge trypsin digestion allows faster processing times, typically on the order of 10–60 min. This paper discusses system design issues, typical performance, and a real life application to cholera toxin (CTx) and staphylococcal enterotoxin B (SEB). Experimental Reagents Bovine serum albumin (BSA), 97.0%, was purchased from Fluka (Buchs, Switzerland). Horse heart myoglobin and iodoacetamide were purchased from Sigma (St. Louis, 1 Abbreviations used: 2D, two-dimensional; SEC, size-exclusion chromatography; CTx, cholera toxin; SEB, staphylococcal enterotoxin B; BSA, bovine serum albumin; ESI, electrospray ionization; BMS, bis(2-mercaptoethyl)sulfone; TIC, total ion current; LOD, limit of detection.
151
MO, USA). Cholera toxin and staphylococcal enterotoxin B were obtained from List Biological Laboratories (Campbell, CA, USA). Sequencing-grade modiWed trypsin was obtained from Promega Benelux (Leiden, The Netherlands). Acetonitrile and calcium chloride were obtained from J.T. Baker (Deventer, The Netherlands). Formic acid and ammonium bicarbonate were from Riedel-de-Haën (Seelze, Germany). Bis(2-mercaptoethyl)sulfone was purchased from Toronto Research Chemicals (North York, ON, Canada). Enzymegrade tris(hydroxylmethyl)aminomethane (Tris) was obtained from Ultrapure, Bethesda Research Laboratories (Gaithersburg, MD, USA). Milli-Q water was obtained from a local Milli-Q Academic system (Millipore, Bedford, MA). All solvents were Wltered and degassed before use. Instrumentation A schematic of the chromatographic setup is given in Fig. 1. The LC-ESI MS unit consisted of a micropump (pump 2) (Intelligent Pump 301M; Flom, Tokyo, Japan), a Luna C18(2) column (100 £ 2.1 mm 3 m; Phenomenex, Bester, Amstelveen, The Netherlands), and a Quattro II mass spectrometer (Micromass, Wythenshawe, Manchester, UK). For a determination of limits of detection a Q-TOF mass spectrometer (Micromass) was used. The LC column was mounted in a column oven kept at 45 °C. The SEC unit consisted of a Lab Alliance pump (pump 1) (Instrument Solutions, Nieuwegein, The Netherlands) coupled to a Biosep 2000 SEC column (3.0 £ 4.6 mm; Phenomenex) in a column oven at 45 °C and an LC 2010 C UV detector set at 214 nm (Shimadzu, ’s Hertogenbosch, The Netherlands). For injection, the SEC unit was equipped with an LC 2010 C autoinjector (Shimadzu). The trypsin cartridge was a Poroszymeimmobilized trypsin digestion cartridge (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). The trapping unit consisted of an LC 2010 C pump (pump 3; Shimadzu) and a C18-ODS guard column (10 £ 2.1 mm; Phenomenex). Sample pretreatment Protein denaturation, disulWde reduction, and thiol derivatization were done by a known protocol [14]. BrieXy, a solution containing 200 L Rapigest SF (0.2% v/v in 50 mM NH4HCO3) and 200 L bis(2-mercaptoethyl)sulfone (2 mg/mL) (BMS) was mixed in an Eppendorf vial and 400 L of a protein solution (100 M) was added. This mixture was incubated for 45 min at 58 °C in the dark, after which the mixture was cooled to room temperature. Then 100 L of iodoacetamide (70 mg/mL) was added and the mixture was placed in a water bath of 37 °C, for 0.5 h. The sample mixture was kept for a maximum of 2 weeks in the refrigerator at 4 °C until use. On-line digestion and analytical separation setup Separation of the denatured, derivatized proteins from the denaturation and derivatization reagents was accom-
152
Determination of denaturated proteins and biotoxins / J. Carol et al. / Anal. Biochem. 346 (2005) 150–157
Fig. 1. Chromatographic setup. The system consist of two automatedswitching six-port valves, two isocratic pumps (pumps 1 and 2), one gradient pump (pump 3), an auto injector, a size-exclusion column (SEC), a UV detector, a trypsin cartridge, a trapping column, an analytical LC column, and a mass spectrometer.
plished by SEC. Typically, 50 L of the denatured, derivatized protein solution was injected and SEC was performed with the trypsin digest buVer (50 mM Tris and 10 mM CaCl2, adjusted to pH 8.0) as the eluent, at a Xow rate of 50 L/min. The UV detector was set at 214 nm. The sample Xow through the SEC column, trypsin digest cartridge, and analytical LC column was established and controlled with the use of two six-port switching valves (Prospekt 1, Spark Holland, Emmen, The Netherlands), as shown in Fig. 1. All components were coupled by blue (0.25mm ID) and red (0.13-mm ID) stripe-coded poly ether ketone tubing (Bester, Amstelveen, The Netherlands). The total analytical procedure encompasses six steps. First, the six-port valve 1 is switched to temporarily transfer all SEC Xow, in digestion buVer 50 L/min, to waste (position 1). Second, valve 1 is switched to load the trypsin cartridge with a selected protein fraction from the SEC column (position 2). Third, valve 1 is switched back to divert the SEC Xow to waste and to allow trypsin digestion to take place during a predetermined stopped Xow period (position 1). Fourth, after this period, the Xow from pump 3 is switched on to
Xush the trypsin cartridge and to trap the digest peptides on the trapping column with a Xow of 50 L/min of digestion buVer (valve 2 in position 1). During the stopped Xow period, the SEC separation is completed. Fifth, valve 1 is switched to position 2, for a desalination wash of the trapping column with water/acetonitrile (95:5 v/v) containing 0.2 v/v-% formic acid and regeneration of the SEC column and the trypsin cartridge. Sixth, valve 2 is switched to transfer the trapped peptides from the trapping column to the LCMS subsystem (position 2). Thus, the LC-MS analysis run completes the overall analytical procedure. The LC gradient was programmed from 100% solvent A (water/acetonitrile, 95:5 v/v with 0.2 v/v-% formic acid) to 80% solvent B (water/ acetonitrile, 5:95 v/v, with 0.2 v/v-% formic acid) in 60 min, at a Xow rate of 0.1 mL/min. The analytical LC system was directly coupled to the ESI interface of a triple-quadrupole MS instrument (Quattro II; Micromass). The Xow rate of the mobile phase, 0.1 mL/min, was compatible with ESI. The mass spectrometer was set to scan from 200 to 1400 Da, scan time of 3.5 s, at a cone voltage of 30 V, a source temperature of 140 °C, and a capillary voltage of 3.7 kV. The ESI nebulizing gas Xow was set at 17 L/h and the drying gas was set at 375 L/h. When a Q-TOF MS was used the system settings chosen were similar to those of the Quattro II MS. Results and discussion Size-exclusion chromatography Separation of a protein mixture in SEC occurs as a function of the average molecular weight of the proteins. Exclusion volumes and derived exclusion times were obtained by injecting a mixture of known proteins directly on the SEC column and using UV detection (214 nm). A continuous Xow of the digest buVer was pumped as the SEC eluent at a Xow rate of 250 L/min. A typical SEC chromatogram is given in Fig. 2, and derived exclusion times are depicted in Fig. 3. The relative standard deviation of the exclusion times of the diVerent proteins was between 0.45 and 1.38% (n D 6). This constitutes a calibration of the SEC subsystem.
Fig. 2. SEC chromatogram of a mixture of three test proteins; the fraction enclosed by the two lines was typically sent to the digest cartridge.
Determination of denaturated proteins and biotoxins / J. Carol et al. / Anal. Biochem. 346 (2005) 150–157
Trapping column
108 107 Blue dextran 2000
MW (Da)
106 Adolase
105
BSA Ovalbumin Myoglobin
104 103
Diazepam Enkephalin
102 101 R2 = 0.9941 0 1.5 2
153
2.5
3
3.5
4
4.5
exclusion volume (mL)
Fig. 3. Calibration graph of exclusion time versus protein molecular weight.
After digestion, the peptides formed were transferred to the trapping column by Xushing the trypsin cartridge with digestion buVer. Generally, peptides have a relatively high aYnity to the C18 material of the trapping column, as compared to the (buVer) salts. Breakthrough volumes were determined by Xushing the trapping column with the LCESI MS eluent used at the beginning of the analytical gradient. Peptide breakthrough occurred at a volume of 1.50 mL. At a Xow of 100 L/min, a 5-min Xush provided suYcient time for desalination. As the Xush volume was well below the breakthrough volume, desalination did not signiWcantly aVect the trapping of the analyte peptides in the present system. LC-MS
Since, with a constant Xow, the exclusion volume is proportional to exclusion time, a fraction with a certain bandwidth around a given average molecular weight (MW) is selected for transfer to the trypsin digestion column. The SEC chromatogram, Fig. 2, shows that the late eluting proteins have a broader peak than the early eluting proteins. As a consequence, a certain Wxed fraction volume passed from the SEC column to the trypsin cartidge represents a more narrow MW range for the highMW proteins than for the low-MW proteins. BSA and myoglobin were used to further investigate the eVect of this MW bandwidth on SEC heart-cutting. In the present system, the volume of the trypsin cartridge is 88 L. From the SEC calibration it is derived for BSA and myoglobin that 88 L of exclusion volume corresponds to MWs of 10 and 7 kDa, respectively. For the toxins studied, SEB (28 kDa) and CTx (A-chain, 29 kDa), the selected exclusion volume is predicted to represent a bandwidth of 8 kDa.
A linear, binary LC gradient of water and acetonitrile was used for the analytical separation of the peptide mixture. Separation starts with elution of the peptides from the trapping column, switched on-line with the LC column, under conditions of the initial eluent composition. The initial eluent composition requires a relatively high concentration of water (95%, v/v), to allow retention of relatively apolar peptides. Acetonitrile (at a minimum of 5% v/v) was employed in the eluent to avoid the formation of gas bubbles during gradient mixing, whereas formic acid (0.2 v-%) was used to prevent secondary adsorption eVects and to provide protons for ESI. A total ion current (TIC) chromatogram was obtained from the digest products by detection with a triple-quadrupole mass spectrometer. The peptide molecular weights were determined from the ESI mass spectra of an LC-MS run by transformation to [M+H]+ ion masses, in a peptide map. The peptide map was then submitted to a Mascot [18] database search.
On-line digestion
Overall setup
To determine the minimum required digestion time, the amount of undigested myoglobin was monitored as a function of time at 5, 10, 20, 30, and 60 min. The peak area of myoglobin without digestion was taken as 100%. It was observed that 70 § 1.5% of the original myoglobin was digested using a 20-min digestion time. This degree of digestion was suYcient for identiWcation by peptide mapping (Mascot score >72, at p < 0.05). Shorter digestion times did not result in identiWcation of myoglobin. That 20 min digestion was appropriate was conWrmed with digestion of BSA, where 30 min digestion time did not produce signiWcant diVerences from 20 min. For comparison, the oV-line (in solution) digestion to a similar endpoint took typically between 4 and 8 h. With the observations made, the minimum digestion time of 20 min was used throughout further experiments; the rate of digestion was suYcient for identiWcation in all cases.
To test the system performance, 10 L of a BSA solution (1 mg/mL) was injected. The TIC chromatogram of that analysis is shown in Fig. 4. BSA digest peptides are clearly resolved and allow derivation of a peptide map. That map gave a positive Mascot hit for BSA, with a protein score of 176 at 58% sequence coverage. A second performance test concerned injection of a mixture of proteins including BSA. The SEC fraction of 60–70 kDa was selected for digestion, whereas the remainder was Xushed to waste. The TIC chromatogram obtained is shown in Fig. 5. The derived peptide map again gave a signiWcant score of BSA (score 137, 56% coverage). The peptides observed were the same as those seen in the analysis of neat BSA. The reproducibility of the performance of the entire system was determined by examination of Wve trypsin digest peptides of BSA (T14, T33, T57, T58, and T82) in multiple analyses (n D 8) and comparison of the variance in reten-
154
Determination of denaturated proteins and biotoxins / J. Carol et al. / Anal. Biochem. 346 (2005) 150–157 23.83
100
27.06
22.30 21.28
35.81
31.05 31.65 17.62
%
33.60 4.79
9.12 41.25 42.53 46.69
Time 50.00
0 10.00
20.00
30.00
40.00
Fig. 4. Total ion chromatogram obtained from the injection of neat (10 L, 20 M) BSA onto the SEC column; the chromatogram is not two dimensional, as it reXects the digestion and analysis of the BSA fraction from SEC. 22.21
100 21.87 18.73
23.91 24.42
21.19 26.80
30.71
28.59
39.81
37.09
%
40.57 2.41
4.02
35.13 41.85 42.95
47.80 44.48 49.84
0 5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
60.00
65.00
Time 70.00
Fig. 5. Total ion chromatogram from the injection of a mixture of proteins (10 L, 25 M of BSA), where the BSA fraction from SEC was transferred online to the digest cartridge; intact BSA was not visible in the chromatographic range displayed.
tion time and abundance. The retention times displayed a relative standard deviation between 1.3 and 1.7%. The standard deviation of the ion abundances was between 13 and 21%. These relatively high standard deviations of the abundances were probably due to variations in the on-line digestion for BSA but did not aVect the identiWcation of the proteins. To compare performance of the overall methodology with diVerent mass spectrometers, the entire chromatographic system was coupled to a Q-TOF mass spectrometer. Chromatograms obtained with the Q-TOF were well comparable to those obtained with the triple quadrupole instrument, and the LC peak shapes in both chromatograms were similar. With the Q-TOF MS the method LOD was 1.14 M for neat BSA and 2.28 M for BSA in
the mixture. This method LOD was one order of magnitude lower than that obtained with the triple-quadrupole instrument, 12 and 18 M, respectively (§1.3 M, n D 3). Toxins Finally, the entire system was used for target compound analysis in two toxin-containing samples. The Wrst target compound was SEB with an average MW of 28.4 kDa. SEB is produced by various strains of Staphylococcus aureus and is considered a potential biological warfare agent. SEB can straightforwardly be identiWed by oV-line sample treatment and LC-MS [19]. Here, the SEC fraction of 25–35 kDa was selected for on-line digestion and chromatography with
Determination of denaturated proteins and biotoxins / J. Carol et al. / Anal. Biochem. 346 (2005) 150–157
A
155
20.34
100
24.42
%
23.57 21.62 25.02 22.72
14.65 4.665.215.98 7.17 6.74 7.51 8.70 10.82 11.93 9.21 13.46 11.25
29.01 26.55
16.60
25.44
19.58 15.92 17.6218.81
28.67 27.99
57
Time 6.00
B
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
ScanES TIC
25.75
100
28.00
29.20 22.90 36.52 20.77 21.78 19.95
26.76 23.51 23.92
17.82 14.97
29.71
18.83
28.09
41.81 30.12 31.14
% 41.30
33.88 32.25 34.90 37.74
39.27 39.78
43.13 43.95
0
Time 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
Fig. 6. Two total ion chromatograms from diVerent samples containing biological toxins. (A) Sample containing SEB (10 L, 25 M); (B) sample containing CTx (10 L, 25 M).
triple-quadrupole MS detection. The TIC chromatogram obtained is shown in Fig. 6A. The peptide map, summarized in Table 1, yielded a Mascot rank 1 match for SEB with a score of 101 and 40% sequence coverage. This provided ample evidence that the sample contained SEB. The second target compound was CTx with A- and Bchain average molecular weights of 29.3 and 11.6 kDa, respectively. CTx is the main toxic product of toxinogenic
strains of Vibrio cholera and the toxin itself is considered a potential biological warfare agent. A molecule of CTx consists of two non-covalently bound units, A(ctive)-chain and B(inding)-chain, which appear in a one-to-Wve ratio. CTx can straightforwardly be identiWed by oV-line sample treatment and LC-MS [20]. Here, the SEC fraction 25– 35 kDa was selected, so only the A-chain was directed toward the digest cartridge. The TIC chromatogram
Table 1 Observed trypsin digest peptides from the sample containing SEB Fraction
Retention time (min)
m/z value detected [M+H]+
m/z value theoretical [M+H]+
Sequence
T1 T3 T4 T7
14.65 22.55 20.34 16.60
1519.73 1070.49 1586.81 950.46
ESQPDPKPDELHK FTGLMENMK VLYDDNHVSAINVK LGNYDNVR
T22
26.55
1278.66
NLLSFDVQTNK
T22-23 T24-25 T25 T26 T29 T35
22.81 21.70 23.66 25.02 24.42 21.53
1518.24 1070.32 1585.34 950.43 950.68 1277.22 1278.32 1404.78 1435.28 1307.08 658.91 1836.26 966.61
1406.76 1436.77 1308.67 659.38 1837.86 966.54
NLLSFDVQTNKK KVTAQELDYLTR VTAQELDYLTR HYLVK LYEFNNSPYTGYIK IEVYLTTK
156
Determination of denaturated proteins and biotoxins / J. Carol et al. / Anal. Biochem. 346 (2005) 150–157
Table 2 Trypsin digest peptides found after Mascot search of the peptide map from the sample containing CTx from V. cholera strain 569B (NCBI CAA41590, gi|48889) Fraction
Retention time (min)
m/z value detected [M+H]+
m/z value theoretical [M+H]+
Sequence
T5 T6 T7 T9 T15 T17 T20 T25
19.95 36.52 33.88 22.80 25.75 26.76 29.20 21.17
845.23 1001.03 1532.79 1449.68 2594.78 1867.74 1029.42 376.20
845.42 1001.42 1532.72 1449.69 2594.22 1867.86 1029.48 376.16
QSGGLMPR GQSEYFDR GTQMNINLYDHAR HDDGYVSTSISLR YYSNLDIAPAADGYGLAGFPPEHR EEPWIHHAPPGCGNAPR FLDEYQSK DEL
obtained is shown in Fig. 6B, whereas the derived peptide map is summarized in Table 2. The Mascot search resulted in rank 1 identiWcation of CTx, with a score of 101 and 40% sequence coverage. This target analysis readily identiWed the presence of CTx. Conclusion A fully automated on-line analytical system was developed for the identiWcation of a size-exclusion-selected protein fraction from a mixture of proteins. The system couples size-exclusion chromatography on-line to a digest cartridge, analytical peptide separation, and MS or MS/ MS detection. The system was used to identify biological toxins by target compound identiWcation. The total analysis time was reduced to 100 min. This comprises 15 min for SEC separation, 20 min for on-line digestion, 5 min for desalination, and 60 min for analytical separation. The system prevents sample handling losses and eliminates tedious sample preparation steps. As a result, this analysis procedure is much faster than oV-line MS-based identiWcation methods for biological warfare agents, which can take up to 2 days. A drawback of the present system setup is that only a single fraction from a complete SEC run can be transferred for digestion and subsequent LC-MS analysis. The remainder of the protein mixture is transferred to waste. In the analysis of MW-limited targets, for example bacterial 60kDa heat shock proteins, this need not be a problem. For more complicated applications, multiple digestion cartridges may be employed to hold fractions during analysis (see, for example [21]). However, this approach is beyond the scope of our present interest. Two toxins, SEB and CTx, were analyzed with the current system and methodology. These toxins were identiWed by database searching, both with a result of 40% sequence coverage. In the near future, the setup will be miniaturized by the incorporation of micro-LC. Miniaturization will allow application of smaller sample volumes and lower LODs, both favorable in case of microorganism identiWcation. The miniaturized system will be employed as a fast and sensitive method for rapid identiWcation of microorganisms.
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