Trace Analysis of Proteins Using Postseparation Solution-Phase

Trace Analysis of Proteins Using Postseparation Solution-Phase Digestion and .... the frequent recalibration of the system and eventually require the ...
0 downloads 0 Views 181KB Size
Download PDF
Anal. Chem. 2007, 79, 1591-1598

Trace Analysis of Proteins Using Postseparation Solution-Phase Digestion and Electrospray Mass Spectrometric Detection of Marker Peptides B. Bruyneel, J. S. Hoos, M. T. Smoluch, H. Lingeman, W. M. A. Niessen, and H. Irth*

Vrije Universiteit Amsterdam, Faculty of Sciences, Department of Analytical Chemistry and Applied Spectroscopy, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

Analytical methodologies for the absolute quantitation of proteins typically include a digest step often using trypsin as the proteolytic enzyme. In the majority of cases, offline and on-line digestion methods are implemented prior to an LC-MS analysis system, requiring a high sequence coverage for unambiguous protein identification. For proteins with a strong overlap in amino acid sequence, e.g., therapeutic proteins and their metabolites, it is essential to separate proteins prior to digestion and the subsequent electrospray mass spectrometry analysis of marker peptides. Here, we present an on-line postcolumn solution-phase digestion methodology that is based on the continuous infusion of the proteolytic enzyme pepsin downstream to the nano C18 reversed-phase column. Proteins are identified based on their retention time in combination with the detection of specific marker peptides formed in the postcolumn digest. The optimization of important parameters such as enzyme concentration, reaction time, and organic modifier concentration is described. We demonstrated that the continuous-flow solution-phase digest method can be coupled on-line to the reversed-phase gradient liquid chromatography separation of proteins. Detection limits obtained for five model proteins, detected as specific marker peptides with m/z values of 300-1000, range from 30 to 90 fmol, with a linear response up to 3 pmol. Quantitative protein analysis currently is an important research topic, judged from the high number of review papers published.1-5 Mass spectrometry (MS) plays a prominent role in this area. Strategies have been developed that allow comparative quantitative proteomics via chemical labeling by isotope-coded affinity tags (ICAT1) or other stable isotope tags. Stable isotope labels can be incorporated in vitro at the protein or peptide level or via in vivo metabolic labeling. This enables detection and relative quantification of up- or down-regulated proteins in complete proteomes of * Corresponding author. E-mail: [email protected]. Fax: +31 20 5987543. (1) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (2) Julka, S.; Regnier, F. J. Proteome Res. 2004, 3, 350-363. (3) Kirkpatrick, D. S.; Gerber, S. A.; Gygi, S. P. Methods 2005, 35, 265-273. (4) Leitner, A.; Lindner, W. J. Chromatogr., B 2004, 813, 1-26. (5) Lill, J. Mass Spectrom. Rev. 2003, 22, 182-194. 10.1021/ac0616761 CCC: $37.00 Published on Web 12/20/2006

© 2007 American Chemical Society

organisms in different states6,7 and/or in relation to the discovery of disease biomarkers.8 Next to relative quantitative protein analysis for protein expression profiling, strategies have been developed for absolute quantitative analysis of proteins9 and peptides.3 A more advanced strategy for comparative quantitative proteomics, based on accurate mass and retention tags, was described by the group of Smith.10,11 Absolute quantification of proteins in biological samples can be important in biomarker studies but also during the development of protein drugs in the pharmaceutical industry. Gerber et al.12 reported the introduction of stable isotope labeled peptides as internal standards during protein digestion, to enable absolute quantification of the proteins using LC-MS-MS in selectedreaction monitoring (SRM) mode. Our group recently reported a multidimensional LC-MS strategy for quantitative bioanalysis of protein drugs in biological matrices.13 The method consists of isolation of the target protein from the biological fluid by immunoaffinity chromatography (IAC), on-line enzymatic digestion of the protein, transfer of the peptides to a solid-phase extraction (SPE) column as an intermediate concentrating and washing step, and finally eluting via a gradient reversed-phase liquid chromatography (RPLC) separation into an electrospray mass spectrometer (ESI-MS) for quantification based on specific marker peptides. Unlike in a global proteomics strategy, where one needs a number of peptides for protein identification, we basically need only one protein-specific marker peptide to perform quantification. It is important to appreciate this difference between targeted quantitative analysis of specific proteins, e.g., protein drugs in biological fluids, and proteomics strategies, where the identification of (6) Krijgsveld, J.; Ketting, R. F.; Mahmoudi, T.; Johansen, J.; Artal-Sanz, M.; Verrijzer, C. P.; Plasterk, R. H.; Heck, A. J. Nat. Biotechnol. 2003, 21, 927931. (7) Washburn, M. P.; Ulaszek, R.; Deciu, C.; Schieltz, D. M.; Yates, J. R., III. Anal. Chem. 2002, 74, 1650-1657. (8) Bischoff, R.; Luider, T. M. J. Chromatogr., B 2004, 803, 27-40. (9) Silva, J. C.; Gorenstein, M. V.; Li, G. Z.; Vissers, J. P.; Geromanos, S. J. Mol. Cell. Proteomics 2006, 5, 144-156. (10) Patwardhan, A. J.; Strittmatter, E. F.; Camp, D. G., II; Smith, R. D.; Pallavicini, M. G. Proteomics 2006, 6, 2903-2915. (11) Qian, W. J.; Monroe, M. E.; Liu, T.; Jacobs, J. M.; Anderson, G. A.; Shen, Y.; Moore, R. J.; Anderson, D. J.; Zhang, R.; Calvano, S. E.; Lowry, S. F.; Xiao, W.; Moldawer, L. L.; Davis, R. W.; Tompkins, R. G.; Camp, D. G., II; Smith, R. D. Mol. Cell. Proteomics 2005, 4, 700-709. (12) Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6940-6945. (13) Hoos, J. S.; Sudergat, H.; Hoelck, J. P.; Stahl, M.; de Vlieger, J. S.; Niessen, W. M.; Lingeman, H.; Irth, H. J. Chromatogr., B 2006, 830, 262-269.

Analytical Chemistry, Vol. 79, No. 4, February 15, 2007 1591

proteins in complex proteomes, protein-protein interactions, and protein expression is being studied. A critical step in our multidimensional LC-MS system is the on-line generation of marker peptides originating from the target protein through proteolytic digestion. Trypsin is the most widely applied enzyme to achieve this in MS-based analytical strategies for protein analysis and proteomics research. Trypsin digestion can be performed in either in-gel format after two-dimensional gel electrophoresis14 or in-solution format for isolated proteins or complete proteomes.15 In this way, it is an essential step in bottomup and shotgun protein characterization strategies based on peptide mass fingerprinting and peptide sequence analysis in combination with database searching.16,17 In our system,13 protein digestion is performed because quantification by LC-MS can be performed more reliably and more sensitively at the peptide level rather than at the intact protein level. For application in multidimensional chromatographic systems, immobilized enzyme reactors (IMER) have been developed, where trypsin is immobilized on either POROS material or macroporous monolithic supports to enable on-line digestion and separations.18,19 IMERs have found application in between the two steps of a two-dimensional LC system, e.g., after immobilized metal-ion affinity chromatography,20 size-exclusion chromatography,21 or immunoaffinity chromatography,13,22 and prior to SPE-RPLC coupled to MS. Alternatively, IMERs have been operated in direct coupling to ESI-MS.23,24 In these systems, IMERs have been used either in stop-flow mode or in flow-through mode. Disadvantages of applications incorporating IMERs include the need to perform digestion at a suitable pH, the reduced digestion efficiency in the presence of high percentages of organic solvents, and the fact that the digestion is relatively slow, compared to the chromatographic time scale. Therefore, the main challenge in these multidimensional approaches is the establishment of suitable conditions between the sample preparation step and the analysis step to ensure high digestion efficiency. Precolumn or direct-infusion on-line digest systems have a main disadvantage in the analysis of samples that contain closely related species of proteins, e.g., a parent protein drug and its metabolites. Any shotgun approach is unable to differentiate between different protein species providing a strong overlap of peptide fragments after digestion. In order to address this issue, we report a postseparation digestion methodology that is based on the separation of protein analytes by liquid chromatography, the postcolumn infusion of a proteolytic enzyme, and the detection (14) Shevchenko, A.; Jensen, O. N.; Podtelejnikov, A. V.; Sagliocco, F.; Wilm, M.; Vorm, O.; Mortensen, P.; Shevchenko, A.; Boucherie, H.; Mann, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14440-14445. (15) Ducret, A.; Van Oostveen, I.; Eng, J. K.; Yates, J. R., III; Aebersold, R. Protein Sci. 1998, 7, 706-719. (16) Delahunty, C.; Yates, J. R., III. Methods 2005, 35, 248-255. (17) Thiede, B.; Hohenwarter, W.; Krah, A.; Mattow, J.; Schmid, M.; Schmidt, F.; Jungblut, P. R. Methods 2005, 35, 237-247. (18) Girelli, A. M.; Mattei, E. J. Chromatogr., B 2005, 819, 3-16. (19) Massolini, G.; Calleri, E. J. Sep. Sci. 2005, 28, 7-21. (20) Riggs, L.; Sioma, C.; Regnier, F. E. J. Chromatogr., A 2001, 924, 359-368. (21) Carol, J.; Gorseling, M. C.; de Jong, C. F.; Lingeman, H.; Kientz, C. E.; van Baar, B. L.; Irth, H. Anal. Biochem. 2005, 346, 150-157. (22) Ji, J.; Chakraborty, A.; Geng, M.; Zhang, X.; Amini, A.; Bina, M.; Regnier, F. J. Chromatogr., B 2000, 745, 197-210. (23) Cooper, J. W.; Chen, J.; Li, Y.; Lee, C. S. Anal. Chem. 2003, 75, 10671074. (24) Krenkova, J.; Bilkova, Z.; Foret, F. J. Sep. Sci. 2005, 28, 1675-1684.

1592

Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

and quantification of peptide markers by ESI-MS. Postcolumn digestion methodologies have recently been described by Slysz and co-workers25,26 using an immobilized trypsin reactor downstream to a C4 reversed-phase separation column. The authors showed the separation of intact proteins followed by ESI-MS detection of peptides produced in the trypsin IMER. By using a postcolumn counter gradient system prior to the IMER they could further improve baseline stability.25 While separation of very similar protein species, such as, for example, protein drugs and their metabolites or protein-drug adducts, using RPLC can be a very difficult task, several methods have been evaluated for this purpose.27 IMERs, in comparison to solution-phase digestion approaches, have the advantage of high enzyme density and, consequently, relatively fast digestion times compared to those of off-line digestion; they may exhibit, however, a long-term instability due to irreversible degradation of enzymes, particularly when placed downstream to a reversed-phase separation method using significant amounts of organic modifiers in the mobile phase. In a postcolumn implementation for quantitative purpose, a changing enzyme activity will require the frequent recalibration of the system and eventually require the replacement of the IMER. Furthermore, IMERs require significant efforts to prepare and characterize the immobilized enzyme supports. In the current paper, we report on the integration of a solutionphase postcolumn digestion method into a RPLC-ESI-MS system. The proteolytic enzymes are continuously infused and mixed with the LC effluent, and selected marker peptides that are representative of the protein to be quantified are detected at their respective m/z values by ESI-MS. We have previously reported postcolumn protease assays where a similar setup has been used to detect protease inhibitors eluting from a RPLC column.28,29 Several other authors have reported the use of solution-phase digestion in various stages of the analytical method.30,31 The solution-phase digestion approach reported here allows the rapid selection and screening of proteolytic enzymes for the specific protein analysis methodology to be developed. When coupled to RPLC for the purpose of protein digestion, the postcolumn reaction system should use proteolytic enzymes that are sufficiently stable and active in aqueous-organic solvents containing acetonitrile or methanol as commonly used in, for instance, a RPLC mobile phase.32 Furthermore, the enzyme should be active under the acidic pH conditions favored for optimum MS ionization. The solution-phase approach essentially allows the implementation and testing of a large variety of proteolytic enzymes without the need to prepare and characterize IMERs. In the current paper, we have chosen pepsin as proteolytic enzyme for postcolumn digestion. Pepsin can be readily applied under acidic conditions but is not frequently applied in proteomics-related (25) Slysz, G. W.; Lewis, D. F.; Schriemer, D. C. J. Proteome Res. 2006, 5, 19591966. (26) Slysz, G. W.; Schriemer, D. C. Anal. Chem. 2005, 77, 1572-1579. (27) Zhou, S. J. Chromatogr., B 2003, 797, 63-90. (28) de Boer, A. R.; Bruyneel, B.; Krabbe, J. G.; Lingeman, H.; Niessen, W. M.; Irth, H. Lab Chip 2005, 5, 1286-1292. (29) de Boer, A. R.; Letzel, T.; van Elswijk, D. A.; Lingeman, H.; Niessen, W. M.; Irth, H. Anal. Chem. 2004, 76, 3155-3161. (30) He, Y.; Zhong, W.; Yeung, E. S. J. Chromatogr., B 2002, 782, 331-341. (31) Okerberg, E.; Shear, J. B. Anal. Chem. 2001, 73, 1610-1613. (32) Russell, W. K.; Park, Z. Y.; Russell, D. H. Anal. Chem. 2001, 73, 26822685.

Figure 1. General setup of the system. P1: Capillary gradient HPLC system 200 nL/min. P2: Infusion pump for pH adjustment 100 nL/ min. P3: Infusion pump for enzyme addition 100 nL/min. 1: Micro mixing union. 2: Fused-silica capillary reactor.

studies because of its limited specificity in protein digestion. It has found some application in the determination of disulfide bonds in proteins33 and in protein conformation studies involving H/D exchange experiments, where an acidic solution is favorable because it reduces the back-exchange of deuterium to the bulk solution.34 In this study, we report the characterization of on-line postcolumn continuous-flow microreactors for the pepsin digestion of target proteins prior to ESI-MS. EXPERIMENTAL SECTION Chemicals and Materials. The model proteins used in this study such as human serum albumin (A9511), transferrin (human siderophilin, T8158), cytochrome c (horse heart, C2506), myoglobin (horse heart, M1882), hemoglobin (human, H7379) as well as the porcine stomach mucosa pepsin (P7000, 632 U/mg solid and 1470 U/mg protein) were purchased from Sigma (St. Louis, MO), except bovine serum albumin (BSA, 05471), which was obtained from Fluka (Buchs, Switzerland). Reserpine and Val-TyrVal were obtained from Sigma (St. Louis, MO). H-(ALA)6-OH and (Gly0)-Met-Enk were from Bachem (Budendorf, Switzerland). Formic acid was from Riedel-de-Hae¨n (Seelze, Germany). LC-MS grade acetonitrile was purchased from Biosolve (Valkenswaard, The Netherlands). Water was produced by a Millipore (Bedford, MA) Milli-Q unit. Instrumentation. The general setup of the system is shown in Figure 1. It consists of an LC Packings (Amsterdam, The Netherlands) Ultimate gradient HPLC (P1 in Figure 1) and a Famos autosampler, a packed microcapillary nano LC column, two microtee assemblies (type P-775, Upchurch Scientific, Oak Harbor, U.S.A.) for mixing an acidic solvent (P2) and the pepsin solution to the column effluent (P3), a transfer capillary (serving as a reaction coil), a homemade 60 mm × 20 µm (uncoated) fusedsilica nanoelectrospray needle, and a Waters (Manchester, U.K.) Q-TOF-2 quadrupole-time-of-flight hybrid instrument equipped with an in-house-built nanoelectrospray probe. The microcapillary nano LC column was a C18 PepMap300 (150 mm × 75 µm i.d., packed with 5 µm reversed-phase particles with 300 Å pores) purchased from Dionex/LC Packings (Amsterdam, The Netherlands). In part of the study, the nano LC column was replaced by a fused-silica capillary (150 mm × 20 µm i.d.). The transfer capillary had a fixed internal diameter of 20 µm, whereas the length was changed to vary the reaction duration. The injection volume was 100 nL in the column-bypass injections and 40 or 20 nL in on-column injections. The identity of some of the proteolytic fragments was confirmed by means of MS-MS. This was performed either on the (33) Gorman, J. J.; Wallis, T. P.; Pitt, J. J. Mass Spectrom. Rev. 2002, 21, 183216. (34) Kaltashov, I. A.; Eyles, S. J. Mass Spectrom. Rev. 2002, 21, 37-71.

Q-TOF-2 or on an LCQ-Deca (Thermo Finnigan, San Jose´, CA), equipped with a similar laboratory-built nanoelectrospray probe. Procedures. Pepsin solutions (0.063-4.0 mg solid per mL) were made in water. In most studies, a concentration of 0.25 mg/ mL was applied. With a molecular weight of pepsin of 34 623 g/mol,35 this would correspond to approximately 7 µmol/L. However, from the specifications of the pepsin used, 1 mg of solid only contains 0.43 mg of protein. Therefore, the actual pepsin concentration in the 0.25 mg/mL solution is 3.1 µmol/L. Other proteins were dissolved in 0.1% aqueous formic acid or in the carrier solvent, which in most cases contained 16% acetonitrile. Stock solutions of 4-10 mg/mL were diluted into dilution series in the carrier solvent, typically in the range between 0.0125 and 4 mg/mL, corresponding to ca. 0.15 to 60 µmol/L. The LC mobile phase or a carrier flow was delivered at 200 nL/min (Figure 1). It was first diluted with 100 nL/min of an aqueous 0.1% formic acid solution. Next, an aqueous solution of pepsin, typically 0.25 mg/mL, was added at a flow rate of 100 nL/ min. The total flow through the fused-silica incubation coil and introduced in the nanoelectrospray interface was 400 nL/min. Most optimization and characterization studies were performed with 16% acetonitrile in the carrier flow, except of course when the influence of the acetonitrile content of the carrier flow was studied. Optimization of experimental parameters was performed for BSA. Two BSA-related peptides were used as marker peptides, i.e., m/z 562 and 620 (cf., Table 1). In HPLC experiments on the 150 mm × 75 µm i.d. C18 PepMap300 column, gradient elution was performed using solvent A consisting of 0.1% aqueous formic acid (pH 2.6), and solvent B consisting of 80% acetonitrile in 0.1% aqueous formic acid. The gradient program was a linear increase of solvent B in solvent A from 5% to 50% in the first 12 min, followed by a linear increase from 50% to 80% up to 35 min, after which the solvent composition was switched back to the initial conditions, and the column was equilibrated for 40 min prior to the next injection. The flow rate was 200 nL/min. Due to the solvent split in the Ultimate, there was a delay time in the gradient of ca. 18 min. The injection volume in on-column experiments was 40 nL in mixture analysis and 20 nL for the calibration curve. RESULTS AND DISCUSSION Setup. The postseparation digest methodology is based on the on-line infusion of a pepsin solution downstream to the separation column. The digestion reaction takes place in a fusedsilica transfer capillary connected to the inlet of the ESI interface. In principle, this setup can simply be achieved by mixing the column effluent with a pepsin solution that is already adjusted to the appropriate pH. Although such a simple setup basically works, we preferred to apply a slightly more complex system. In our system (Figure 1), the column effluent is first 2:1 diluted with an acidic aqueous solution (0.1% formic acid, pH 2.6) Subsequently, a neutral pepsin solution is added to start the digestion. Pepsin shows rapid proteolytic activity in acidic solution but is almost inactive in neutral solution. Our two-step mixing system greatly reduces the autodigestion of pepsin and also results in a reduced background interference and chemical noise in the nanoelectro(35) http://www.expasy.org/uniprot/P00791.

Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

1593

Table 1. Identification of Proteolytic Fragments by Pepsin Digestion of Bovine Serum Albumin and Myoglobina Bovine Serum Albumin MKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPF DEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEP ERNECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRHPYFYAPELLYY ANKYNGVFQECCQAEDKGACLLPKIETMREKVLASSARQRLRCASIQKFGERALKAWSVA RLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKE CCDKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRR HPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEK LGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLIL NRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLP DTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVVSTQTALA proteolytic fragment detected at m/z 468.6 (6+) 562.3 (5+)* 702.4 (4+) 936.3 (3+) 1403.7 (2+)* 409.0 (4+) 545.0 (3+) 817.0 (2+) 449.3 (4+) 598.7 (3+)* 897.5 (2+)* 344.7 (2+)* 366.2 (2+)* 696.1 (3+)* 465.3 (2+) 620.1 (3+)*

DTHKSEIAHRFKDLGEEHFKGLVL

ARRHPYFYAPELL WSVARLSQKFPKAEF VEVTKL WSVARL AEDKDVCKNYQEAKDAFL IVRYTRKVPQVSTPTL Myoglobin from Horse Heart GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEA EMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLE FISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG proteolytic fragment GLSDGEWQQVLNVWGKVEADIAGHGQEVL IRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVL TALGGILKKKGHHEAELKPLAQSHATKHKIPIKY LEF FRNDIAAKYKELGFQG

detected at m/z 1045.9 (3+)* 1568.4 (2+)* 1163.7 (4+) 1033.9 (4+)* 619.7 (3+)* 465.0 (4+)*

a Fragments indicated in bold were detected. The sequence of the ions indicated with an asterisk (*) was confirmed by MS-MS on either an LCQ ion trap instrument or on the Q-TOF-2 instrument. Other ions were identified by peptide mass fingerprinting.

spray mass spectrometric detection. In addition, the 1:1 (final) dilution also provides a 50% reduction of the organic solvent content in the reaction mixture. Optimization of Analytical Parameters. In order to characterize the postcolumn continuous-flow microreactor, the influence of a number of experimental parameters was studied. These studies were performed in column-bypass mode (flow injection mode). Injections of 100 nL of a solution of bovine serum albumin (BSA) as model protein were made directly into the carrier stream (16% acetonitrile in 0.1% aqueous formic acid). Figure 2 shows the mass spectra obtained for BSA injection in the presence and absence of pepsin in the postcolumn reagent. The BSA envelope (Figure 2, inset) completely disappeared when pepsin was present, indicating a high turnover rate under the conditions chosen. The peptides obtained can serve as markers for the quantification of the original protein concentration. In most cases, we selected two different BSA-derived marker peptides, one with m/z 620 and one with m/z 562 (cf., Table 1, see below). The first peptide generally provides a slightly lower detection limit than the second. The optimization was performed in the order reported. Optimum values 1594 Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

Figure 2. Mass spectrum of in-flow pepsin-digested bovine serum albumin. The inset shows the spectrum of undigested BSA. Background ions originating from the pepsin solution are indicated with 0. Digest fragments from BSA are marked with +. The sequence of ions marked with an asterisk (/) are digest fragments confirmed by MSMS on either an LCQ ion trap instrument or a Q-TOF-2 instrument.

Table 2. Average (n ) 4) S/N Values and Standard Deviations of Selected Reference Compounds in the Absence (S/N - Pepsin) and Presence (S/N + Pepsin) under Optimum Reaction Conditionsa concn (ng/mL)

S/N -pepsin

S/N +pepsin

F

reserpine (m/z ) 609.7)

750 375 150

27.4 ( 1.9 15.3 ( 1.1 8.2 ( 0.3

7.9 ( 1.4 3.9 ( 0.5 1.9 ( 0.3

4.0 4.3 3.7

H-(ALA)6-OH (m/z ) 445.3)

650 325 130

58.9 ( 3.7 31.6 ( 1.4 13.1 ( 0.6

15.4 ( 0.9 7.8 ( 0.7 3.3 ( 0.2

3.8 4.1 4.0

(Gly0)-Met-Enk (m/z ) 631.3)

550 275 110

13.0 ( 1.0 7.1 ( 0.1 3.0 ( 0.4

6.7 ( 0.2 3.9 ( 0.4 2.1 ( 0.3

1.9 1.9 1.4

Val-Tyr-Val (m/z ) 380.3)

500 250 100

33.2 ( 2.2 18.5 ( 0.3 20.2 ( 1.2

12.4 ( 0.7 5.4 ( 0.3 5.9 ( 0.5

2.9 3.3 3.1

compound

a

F is defined as (average S/N - pepsin)/(average S/N + pepsin).

Figure 3. Influence of the pepsin concentration on signal-to-noise values. Analyte, BSA; marker peptide, m/z 562.

from one experiment were used for subsequent optimization experiments. The digestion time was varied by the use of different lengths of the 20 µm i.d. transfer capillary (between 32 and 127 cm, leading at the applied flow rate of 400 nL/min to a reaction time between 15 and 60 s). The best response and signal-to-noise ratio (S/N) for the BSA marker peptides was obtained at a reaction time of 30 s. While a linear increase of the product peptide concentration can be expected as function of the reaction time, the lower response at higher digestion times can probably be attributed to further cleavage of the selected marker peptides by pepsin into smaller peptides. In all further studies, a reaction time of 30 s was applied, which means a 64 cm × 20 µm i.d. transfer capillary. In principle, the influence of the temperature on the digestion efficiency could have been studied. Optimum reaction temperatures for protease IMERs have been reported that are significantly higher than ambient temperature.25,36 However, operating the microreactor at elevated temperatures would result in a more complex and more vulnerable system. As the microreactor is directly coupled to the nanoelectrospray needle, we anticipated potential problems in the mechanical stability of such a setup. Therefore, we decided to perform our measurements at room temperature only. Because the current optimum reaction time is already very short, there also appears not to be any need to speed up the digestion by increasing the temperature. The influence of the pepsin concentration on the response and signal-to-noise (S/N) was evaluated as well. Concentrations of dissolved pepsin between 0.75 and 48 µmol/L were tested for BSA concentrations in the range of 0.2 to 60 µmol/L. One can expect that increasing pepsin concentrations initially result in an increased conversion of the protein analyte to marker peptides. However, too high pepsin concentrations will lead to an increased background noise generated by pepsin constituents and its autodigestion products. Figure 3 shows S/N values as a function of the pepsin concentration. An increasing pepsin concentration leads (36) Visser, N. F. C.; Lingeman, H.; Li, K. W.; Irth, H. Chromatographia 2005, 61, 433-442.

to an increase of the S/N values up to an optimum value at 3.1 µmol/L. Repeated optimization experiments resulted in the same optimum concentration of enzyme. A further increase of the pepsin concentration results in decreasing S/N values due to a combination of increasing noise and degradation of the marker peptides. In the pepsin background spectrum, a number of m/z values were frequently observed, e.g., m/z 325.15, 343.17, 474.27, 490.23, and 685.30. Product ion MS-MS spectra of these single-charge precursor ions suggest that these peaks are due to sugar-like structures, as indicated by (repetitive) neutral losses of 162 and 180 Da. No further attempts were made to identify the compounds responsible for the background, as they are most likely impurities originating from the pepsin preparation. Next to influencing the concentration of marker peptides formed in the digest reaction, the presence of the proteolytic enzyme in the digest solution entering the mass spectrometer may also have a profound effect on ionization efficiency. In order to investigate ion suppression effects, we have determined the S/N values (n ) 4) of a number of reference compounds in the presence and absence of pepsin at the optimum concentration found in earlier optimization experiments (see Table 2). The presence of pepsin results in a decrease of the S/N values by a factor of 2-4. The decrease of S/N values was constant at the concentration range studied. Although the resulting increase in detection limits may be critical for certain applications, it has to be weighted against a high degree of freedom in the choice of the proteolytic enzyme allowing the fast screening of proteolytic enzymes that are ideally suited to produce marker peptides for the protein analyte(s) to be investigated. An important issue of a postcolumn continuous-flow microreactor is its ability to operate at typical solvent compositions required to perform RPLC separations of proteins. With enzymatic reactions, the organic modifier content of the mobile phase generally is the most important issue, unless the presence of a specific cofactor is required, which is not the case with pepsin. We have evaluated the response and S/N of the BSA marker peptides in the continuous-flow microreactor with 4%, 16%, 40%, and 60% acetonitrile in the carrier flow and in the BSA sample Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

1595

Table 4. Minimum Detectable Quantitiesa and Upper Limit of Linearity Tested in the Column-Bypass Continuous-Flow Pepsin Digestion Microreactorb protein

LOD

upper linearity limit tested

BSA

45 fmol (620.1)

e3 pmol (562.3) e1.5 pmol (620.1)

HSA

45 fmol (458.3)

e1.5 pmol (610.7) e3 pmol (458.3)

transferrin

60 fmol (560.9)

e1.4 pmol (560.9) e1.4 pmol (453.3)

cytochrome c

90 fmol (322.9)

e1 pmol (322.9) e1 pmol (483.8)

myoglobin

30 fmol (465.0 and 619.7)

e3 pmol (619.7) e1.5 pmol (465.0)

a LOD, S/N ) 3, m/z value of marker peptide indicated between brackets. b 30 s reaction time, room temperature, 0.25 mg/mL pepsin, 16% acetonitrile in carrier stream.

Figure 4. Influence of the acetonitrile content on detection sensitivity of BSA peptide fragment with m/z 562 at two different protein amounts. (O) injection of 1.5 pmol of BSA, (9) injection of 75 fmol of BSA. Response values are depicted as relative values calculated as (signal intensity × 100)/(maximum signal intensity). Table 3. Identity of Specific Marker Peptides Obtained by Pepsin Digestion of the Model Proteins Applied in This Studya protein bovine serum albumin

m/z charge value state

amino acid sequence

562.3

5+

DTHKSEIAHRFKDLGEEHFKGLVL

620.1

3+

IVRYTRKVPQVSTPTL

myoglobin

619.7 465.0

3+ 4+

FRNDIAAKYKELGFQG

hemoglobin

624.2

6+

VTLAAHLPAEFTPAVHASLDKFLAS VSTVLTSKYR

935.8

4+

human serum 610.7 albumin 458.3

3+

cytochrome c

322.9 483.8

3+ 2+

YLKKATNE

transferrin

560.9 453.3

5+ 1+

not identified not identified

LVRYTKKVPQVSTPTL

4+

a The identity of these marker peptides was confirmed by product ion MS-MS studies on either an LCQ ion trap or a Q-TOF instrument.

solution. The results are shown in Figure 4 for two different BSA concentrations and using m/z 562 as the marker peptide. Given the dilution in the microreactor, these acetonitrile concentrations correspond to 2-30% acetonitrile in the continuous-flow microreactor. The effect was studied in a range of BSA concentrations (0.2-15 µmol/L). Higher acetonitrile concentrations could not be tested due to the limited solubility of BSA at an acetonitrile content higher than 60%. It should also be noted that changes in the acetonitrile content influenced the stability of the electrospray performance. Some optimization of needle voltage and position had to be performed. We found that increasing acetonitrile concentrations lead to a sensitivity loss of approximately a factor 1596 Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

Figure 5. Flow injection response for the injection of different BSA concentrations. (A) Response for marker peptide with m/z 620.2 (B) Response for marker peptide with m/z 562.2. (C) Total ion current signal. Duplicate injections, with the amount of protein injected being indicated at the peak maxima.

of 2 when compared to the optimum response at 4%. The loss in sensitivity stabilized at higher acetonitrile concentrations, probably due to improved electrospray performance under these conditions. Peptide Identification. Although not especially important for our application, i.e., the generation of a protein-specific marker peptide to enable absolute quantification, some attention was also paid to the type of proteolytic fragments formed. The specificity of the marker peptide can only be decided upon when its identity is known. Pepsin is known to cleave at the C-terminal side of the amino acids Ala, Leu, Trp, and Phe,37 whereas cleavages at Met are frequently observed as well. Cleavage at Leu and Phe in particular could be confirmed by detailed identification of peptides from BSA and myoglobin. The peptides obtained are listed in Table 1. It is also known that pepsin digestion results in a relatively high rate of cleavages at other sites than specified and that miscleavages (no cleavage observed although a cleavage is expected) occur relatively often as well. The peptides observed (37) Marie, G.; Serani, L.; Laprevote, O. Anal. Chem. 2000, 72, 5423-5430.

Figure 6. Separation of a mixture of five of our model proteins. (A-E) Extracted ion currents of identified marker peptides for hemoglobin (A), myoglobin (B), human serum albumin (C), transferrin (D), and cytochrome c (E). (F) Sum of extracted ion currents A-E. (G) Total ion current chromatogram.

for some of our model proteins were searched against their known amino acid sequence. The amino acid sequence found in this way could often be confirmed by further MS-MS studies using either an ion trap or a Q-TOF instrument. In most cases, the m/z values of the peptides observed could be correlated to specific peptide fragments, as is shown for BSA and horse heart myoglobin in Table 3. For some proteins, peptide fragments with similar m/z values were observed, indicating the importance of making an adequate selection of the marker peptide used in the quantitative study. In real-life studies, the selection of appropriate marker peptides is also influenced by the matrix in which the target protein must be analyzed and the specificity of the sample pretreatment method applied to isolate the target protein from its biological matrix. The identity of the marker peptides used for the model proteins applied in this study is summarized in Table 3. Analytical Performance in the Flow Injection Mode. In order to assess the quantitative performance of the system in the column-bypass setup, the minimum detectable quantities and linearity were evaluated for five model proteins (bovine and human serum albumin, transferrin, cytochrome c, and myoglobin). Calibration curves were constructed using the previously optimized reaction conditions in the concentration range of typically between 50-100 fmol and 3 pmol injected (100 nL injection volume). The results in terms of limit of detection (LOD, S/N of 3) and the highest concentration still providing good linearity are

summarized in Table 4. A representative flow injection trace is shown in Figure 5 for the on-line solution-phase digestion of BSA. The detection limits obtained with the current method are in the same order of magnitude as those reported by Slysz et al.25 for the optimized postcolumn IMER system, indicating that the background noise caused by the solution-phase digest approach is not hampering the quantitative trace analysis of proteins. On-Line Coupling to RPLC of Intact Proteins. Finally, the postcolumn microreactor system was tested in combination with RPLC analysis of proteins on a nano LC C18 PepMap300 column (150 mm × 75 µm i.d., 5 µm particles). A typical chromatogram for the separation of a mixture of five of our model proteins (hemoglobin, myoglobin, human serum albumin, cytochrome c, and transferrin) is shown in Figure 6. Adequate separation and detection of the five proteins was achieved in this setup (0.5-2.6 pmol of protein injected). The separation was achieved using gradient elution involving a solvent gradient program from 4% to 64% acetonitrile in the LC mobile phase. Despite the changing acetonitrile concentration, good stability of the nanoelectrospray performance and the pepsin digestion could be achieved. Some series of experiments were performed in more than 17 h of continuous and unattended operation. Although the postcolumn microreactor setup contributed to external band broadening of the chromatographic peaks, the effects were limited because relatively symmetric peaks were observed. Due to the slow diffusion of large molecules such as proteins, the chromatographic Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

1597

separation of proteins is generally difficult to perform with high efficiency. For three model proteins, calibration curves were constructed in this on-line LC-postcolumn microreactor-nano ESI-MS setup. Cytochrome c, myoglobin, and BSA showed a linear response in the range between 0.16 and 4.8 pmol injected, 0.2 and 3.5 pmol injected, and 0.1 and 1.8 pmol injected, respectively. In these experiments, our focus was on the lower concentrations. Therefore, the linearity was not tested at higher concentrations. The detection limits (S/N of 3) determined in this way were 3-6 µmol/ L. Obviously, lower concentration detection limits could have been achieved by performing on-line preconcentration using a solidphase extraction approach, at least as long as the mass loadability of the column is not exceeded. CONCLUSION A postcolumn continuous-flow microreactor for the enzymatic digestion of intact proteins by pepsin was evaluated and applied to RPLC chromatographic separation of protein mixtures. Pepsin can be used under acidic solvent conditions to rapidly (within 1 min) digest target proteins in the microreactor. Whereas methanol provides significant reduction of the pepsin enzymatic activity already at low concentrations (10% in the microreactor), the presence of acetonitrile in the microreactor can be tolerated up to about 30%. Given the dilution of the LC column effluent prior to the reaction, this means that an acetonitrile content of the mobile phase up to 60% can be used without significant deterioration of the digestion efficiency of the postcolumn microreactor. The presence of pepsin and its autodigestion products results in a decrease of signal-to-noise values for selected reference compounds by a factor of 2-4.

1598

Analytical Chemistry, Vol. 79, No. 4, February 15, 2007

The potential of pepsin digestion was investigated in our attempts to find alternative ways to generate specific marker peptides in the targeted quantitative bioanalysis of protein drugs in biological fluids. From a protein identification point of view, pepsin digestion is not as easy to predict as, for instance, trypsin digestion. However, for our purpose in quantitative bioanalysis the reproducible formation of a specific marker peptide is required rather than a peptide map that can be searched against a protein database. Therefore, the performance of the postcolumn microreactor was also tested for various model proteins over a concentration range of typically 2 orders of magnitude wide rather than just at one protein concentration. In this way, we could assess whether the protein concentration influences the speed and reproducibility of the digestion. Good quantitative results have been obtained. This same experimental setup could be applied with other proteolytic enzymes, for example pronase, under the condition that the speed of digestion and their compatibility with typical chromatographic solvent conditions is appropriate. The solutionphase approach is ideally suited to rapidly evaluate the applicability of proteolytic enzymes without the need of immobilization and long-term stability testing. We foresee applications particularly in those areas where analysis of similar protein species are encountered, for example, in the metabolic profiling of therapeutic proteins or the analysis of protein-drug adducts.

Received for review September 6, 2006. Accepted October 30, 2006. AC0616761