An On-Line Protein Digestion Device for Rapid Peptide Mapping by

Anal. Chem. 2000, 72, 5423-5430

An On-Line Protein Digestion Device for Rapid Peptide Mapping by Electrospray Mass Spectrometry Ge´rald Marie, Laurent Serani, and Olivier Lapre´vote*

Laboratoire de Spectrome´ trie de Masse, Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif sur Yvette Cedex, France

A simple laboratory-constructed device has been developed for fast on-line protein digestion followed by peptide mapping by use of electrospray mass spectrometry. Taking advantage of its nonsolubility properties at nearneutral pH values, pepsin could be nonpermanently attached to the PEEK tube commonly employed as transfer capillary between the syringe and the electrospray ion source. After optimization of experimental conditions such as pH, solvent, and exposure time, efficient digestion of several model proteins of molecular weights between 14 000 and 66 000, some having disulfide bridges, was successfully carried out. This technique provided reliable and reproducible sequence information by means of C-terminal-specific cleavages of aromatic and hydrophobic residues. As an application, protein identification could be achieved using a protein database search software. In recent years, considerable progress has been made toward rapid identification of unknown or posttranslationally modified proteins by peptide mapping using either electrospray ionization1,2 or matrix-assisted laser desorption/ionization mass spectrometry.3,4 Protein sequence information can now be routinely obtained by several ways. Apart from an elegant method based on the combination of capillary isoelectric focusing and Fourier transform ion cyclotron resonance mass spectrometry described recently by Jensen et al.,5 most of the published works related to proteomics use in situ digestion techniques in 2-D electrophoresis gels prior to mass spectrometric analysis.6-9 An alternative method for digesting proteins consists of constructing a solid-phase column in which protease is immobilized. This could be achieved * Corresponding author: (fax) (33) 169 077 247; (e-mail) [email protected]. (1) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (2) Figeys, D.; Aebersold, R. Anal. Chem. 1998, 70, 3721-3727. (3) Yates, J. R., III. J. Mass Spectrom. 1998, 33, 1-19. (4) Ekstro ¨m, S.; O ¨ nnerfjord, P.; Nilsson, J.; Bengtsson, M.; Laurell, T.; MarkoVarga, G. Anal. Chem. 2000, 72, 286-293. (5) Jensen, P. K.; Pasˇa-Tolic´, L.; Anderson, G. A.; Horner, J. A.; Lipton, M. S.; Bruce, J. E.; Smith, R. D. Anal. Chem. 1999, 71, 2076-2084. (6) Hellman, U.; Wernstedt, C.; Gonez, J.; Heldin, C. H. Anal. Biochem. 1995, 224, 451-455. (7) Davies, M. T.; Lee, T. D. J. Am. Soc. Mass Spectrom. 1998, 9, 194-201. (8) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850-858. (9) Figeys, D.; Ducret, A.; Yates, J. R., III; Aebersold, R. Nat. Biotechnol. 1996, 14, 1579-1583. 10.1021/ac000421z CCC: $19.00 Published on Web 10/05/2000

© 2000 American Chemical Society

by using agarose10 or controlled-pore glass.11,12 Following this idea, Davies et al.13 developed a microscale immobilized trypsin reactor using a macroporous silica support. This technique requires chemical modification of the solid-phase prior to trypsin coupling. From a practical point of view, some improvements may be needed for achieving both mass analysis of intact proteins and identification of peptide digests without changing the injection line. Similarly, switching rapidly from a digestion experiment to a protein mass measurement by using the same introduction line could present some advantages over other procedures. To gain such a versatility, all chemical derivatizations of the solid support, the protease, or the sample protein should be avoided. Finally, the digestion device should be easily adapted to different amounts of sample in order to limit the dead volume effects and to optimize the enzyme/protein ratio. In the present work, we propose to immobilize porcine gastric mucosa pepsin on the inner wall of the introduction capillary of an electrospray ion source (simple red PEEK tube) without any previous chemical reaction, using its nonsolubility properties at near-neutral pH values. The paper describes the construction and the development of this easy on-line digestion protein device combined with an electrospray ionization mass spectrometer. Experimental conditions have been optimized to allow model proteins, with or without disulfide bridges, to be digested. Mass spectrometric analysis of pepsic peptides provides a series of m/z values corresponding to specific cleavages in most cases. The use of a protein database search software allows identification of the protein from which the peptides are generated. The easy and rapid setting-up of this technique makes it very useful for sequencing and identifying unknown or posttranslationally modified proteins. EXPERIMENTAL SECTION Materials. PEEK tubing (red, 130-µm inner diameter) was purchased from Interchim (Montluc¸ on, France). Porcine gastric mucosa pepsin was purchased from Boehringer (Mannheim, Germany). Horse heart myoglobin, bovine hemoglobin, bovine (10) Cobb, K. A.; Novotny, M. Anal. Chem. 1989, 61, 2226-2231. (11) Stachowiak, K.; Wilder, C.; Vestal, M. L.; Dyckes, D. F. J. Am. Chem. Soc. 1988, 110, 1758-1765. (12) Voyksner, R. D.; Chen, D. C.; Swaisgood, H. E. Anal. Biochem. 1990, 188, 72-81. (13) Davies, M. T.; Lee, T. D.; Ronk, M.; Hefta, S. A. Anal. Biochem. 1995, 224, 235-244.

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Figure 1. Mass spectra of a myoglobin solution (water/methanol/acetic acid, 50:50:1, v/v/v) delivered through the pepsin line into the electrospray ion source, recorded at Vsc - Vs values of (a) 36, (b) 84, (c) 132, and (d) 180 V.

serum albumin, hen egg white lysozyme, and DL-dithiothreitol were purchased from Sigma (St. Quentin Fallavier, France). Acetic and formic acids (Merck, Chelles, France), methanol (Prolabo, Fontenay-sous-Bois, France), and ammonium bicarbonate (Aldrich) were used without any further purification. Disulfide Bridge Reduction. Lysozyme and albumin were reduced for 30 min using 26 equiv of DL-dithiothreitol/cysteine at 37 °C under argon atmosphere. On-Line Digestion. A 1-m-long PEEK tube was loaded with 26 µL of a 20 µM solution of pepsin in 2% formic acid, pH 1. After an immobilization time of 10 min, the line was washed with 2 mL of distilled water, corresponding to 80 times the dead volume. The line was thus ready for use. Proteins (10-20 µM in either 5424 Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

H2O/MeOH/AcOH, 50:50:1, v/v/v, or 0.5% aqueous acetic acid) were either directly delivered through the PEEK tube into the electrospray ion source or incubated in the capillary for a predefined time before being pushed into the ion source with 26 µL of aqueous or aqueous methanolic acidic solvents. Under these conditions, no pepsin autolytic peptide was found. The same PEEK tube can be used several times for on-line digestion; the line is regenerated by washing it with a strongly acidic solution. Mass Spectrometry. Experiments were performed by using a Zabspec/T mass spectrometer (Micromass, Manchester, U.K.)14 equipped with an electrospray ionization source. They were carried (14) Scrivens, J. H.; Rollins, K.; Jennings, R. K. C.; Bordoli, R. S.; Bateman, R. H. Rapid Commun. Mass Spectrom. 1992, 6, 272-277.

Figure 2. (a) Amino acid sequence of horse heart myoglobin. (b) Measured and calculated average masses of peptides issued from direct injection of a myoglobin solution through the digestion line.

out at a skimmer voltage (Vs) of 4 kV, and the sampling cone potential (Vsc), controlled by the operator, was adjusted to attain Vsc - Vs values comprised between 0 and 240 V. The temperature of the source was held at 80 °C. The mass spectrometer was scanned over the m/z range of interest, and the mass scale was calibrated by injecting a solution of sodium iodide (Aldrich). Proteolysis products were delivered into the electrospray ion source by means of a syringe-pump PHD 2000 (Harvard Apparatus, Les Ulis, France) at a flow rate of 5 µL/min. MS-Fit Software. Lists of proteolytic peptide monoisotopic masses were used to match possible proteins using the MS-Fit software, part of the “Protein Prospector” program (1995-1999, The Regents of the University of California), created by Karl Clauser and Peter Baker, available either at the Internet site of the University of California San Francisco (http://prospector. ucsf.edu) or at the joint Ludwig Institute for Cancer Research & the University College London web site in the United Kingdom (http://falcon.ludwig.ucl.ac.uk). Parameters used for searches are described and discussed in the following section. RESULTS AND DISCUSSION Experimental Condition Optimization. Pepsin coating on the inner wall of the capillary was applied by introducing an acidic pepsin solution followed by repeated washing with distilled water. Subsequently, an aqueous methanolic acidic solution of myoglobin was delivered through the line into the electrospray source. Figure 1 shows four spectra recorded respectively at Vsc - Vs values of 36, 84, 132, and 180 V. It is noteworthy that an increase of the Vsc - Vs value is accompanied by a reduction of the ion charge states. A deconvolution of the mass spectra allows an unambiguous identification of proteolytic peptides (Figure 2). A slight mass shift is noted for higher mass species (1 unit out of 10 000). These peptides are obtained by cleavages at the C-terminal side of hydrophobic and aromatic residues, demonstrating the specificity of pepsin under these conditions. By varying the exposure time of myoglobin to in-capillary pepsin from 0 to 2 h, mass spectra do not exhibit any notable difference (Figure 3). Apart from the peak intensities, one can thus observe that 2-h digestion is not more efficient than the 5 min required for the solution transfer within the capillary. This

could be explained by enzyme denaturation in the presence of methanol from the solvent mixture. Whereas, according to Dunn and Fink,15 methanol has no deleterious activity on pepsin at low temperature, Line and co-workers16 showed that pepsin, covalently attached on glass, presented a significant activity toward proteins in aqueous solution but was totally inactive in the presence of methanol. This consideration led us to suppress the use of methanol, despite the initial requirement of protein denaturation prior to digestion. Mass spectra of myoglobin proteolyzates were hence performed in 0.5% aqueous acetic acid and recorded after 1 and 2 h (Figure 4). These spectra are to be compared with those shown in Figure 3 where the experimental condition difference lies only in the nature of the employed solvent. Thus, one can clearly see that the digestion is much more efficient in the absence of methanol. The acid denaturation of myoglobin seems to be sufficient to achieve an efficient digestion. Peptide Mapping. Mass spectrum analysis of the 1-h pepsin digestion of myoglobin (Figure 4a) allowed the list of the resulting proteolytic peptides to be drawn up (Table 1). The monoisotopic masses were deduced from the singly and doubly protonated species which were obtained with their highest relative abundances at a Vsc - Vs value of 55 V. The peptides of molecular weight exceeding 2500 were recognized by deconvolution of the high-mass range of the mass spectrum. Their average masses are indicated separately in Table 1. Eleven out of the 14 identified peptides corresponded to the cleavages of the C-terminal side of the following amino acids: alanine, leucine, tryptophan, and phenylalanine. These results allowed us to cover up to 83% of the myoglobin sequence. Bovine hemoglobin is a heterotetrameric complex comprising two R-chains (141 residues, 15 053.2 Da) and two β-chains (145 residues, 15 954.5 Da). A solution of this protein was digested on-line for 4 h and then delivered into the electrospray ion source by pushing it with a 0.5% acetic acid solution. The corresponding mass spectrum is presented in Figure 5. A longer digestion time than in the case of myoglobin was required, possibly due to the higher order structure of the studied system. The series of (15) Dunn, B. M.; Fink, A. L. Biochemistry 1984, 23, 5241-5247. (16) Line, W. F.; Kwong, A.; Weetall, H. H. Biochim. Biophys. Acta 1971, 242, 194-202.

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Figure 3. Mass spectra of a water/methanol/acetic acid solution of myoglobin exposed to in-capillary pepsin for (a) 5, (b) 20, (c) 40, (d) 60, and (e) 120 min. Vsc - Vs was set at 36 V.

peptides, identified either directly in the mass spectrum or by deconvolution, is summed up in Table 2. The presence of two different chains in solution did not appear to be a limiting factor for the peptide mass determination: 8 out of the 16 identified peptides were attributed to the R-chain, the 8 others issuing from the β-globin. Peaks at m/z values of 429.3, 903.6, and 910.6 could have several origins, an ambiguity that could be solved by performing MS/MS experiments. As in the previous case of myoglobin, the site specificity of on-line pepsin digestion was shown to be efficient enough for reliable peptide identification. Hen egg white lysozyme, including four disulfide bridges, was reduced before being injected through the line into the ion source. The resulting mass spectra illustrated the complete conversion 5426 Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

of cystine to cysteine as the average calculated masses of nonreduced and reduced lysozyme are 14 305.2 and 14 313.2 Da, respectively, the measured mass being 14 314.9 Da (Figure 6a and b). Another aliquot of reduced lysozyme was introduced into the digestion line and left 1 h before being delivered into the mass spectrometer for analysis (Figure 6c). The attribution of the peaks corresponding to the singly and doubly charged ions provided a list of proteolytic peptides (Table 3). All of them corresponded to expected cleavages. Thus, 93% of the lysozyme amino acid sequence was recovered. No peak could be attributed to any disulfide bridge recombination: as a consequence, a chemical protection of cysteine, as S-alkylation with iodoacetamide, for example, does not appear to be necessary prior to digestion.

Figure 4. Mass spectra of a 0.5% aqueous acetic acid solution of myoglobin recorded after on-line digestion times of (a) 60 and (b) 120 min. Peak numbers and charge-state values correspond to the peptides listed in Table 1. Table 1. List of Peptides Issued from an In-Capillary 1-h Digestion of Myoglobin origin

peaka

peptide

sequence

Mmeas (Mcalc), Da

mass spectrum (monoisotopic masses)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

135-137 30-32 8-11 1-7 20-29 1-11 56-69 14-29 138-153 12-29 30-55 70-106 33-69 30-69

(A)LEL (L)IRL (W)QQVL ()GLSDGEW (A)DIAGHGQEVL ()GLSDGEWQQVL (M)KASEDLKKHGTVVL (V)WGKVEADIAGHGQEVL (L)FRNDIAAKYKELGFQG (L)NVWGKVEADIAGHGQEVL (L)IRLFTGHPETLEKFDKFHLKTEAEM (L)TALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEF (L)FTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVL (L)IRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVL

373.0 (373.2) 400.1 (400.3) 486.2 (486.3) 762.3 (762.3) 1037.4 (1037.5) 1230.5 (1230.6) 1523.6 (1523.8) 1707.7 (1707.9) 1855.7 (1856.0) 1920.7 (1921.0) 3146.0 (3146.7) 4133.3 (4133.9) 4269.6 (4270.9) 4652.5 (4653.4)

deconvoluted spectrum (average masses)

a

The peak numbers indicated in this table are reproduced in the mass spectrum shown in Figure 4a.

Figure 5. Mass spectrum of a 0.5% acetic acid solution of hemoglobin exposed to in-capillary pepsin for 4 h. Peak numbers and charge-state values correspond to the peptides listed in Table 2.

Moreover, the presence of residual DL-dithiothreitol in the injected solution has no deleterious effect on pepsin at this pH value. Under its monomeric form, bovine serum albumin is a 66-kDa protein that comprises 583 residues and 17 disulfide bridges. After reduction of the protein, the solution was injected into the line for 1-h digestion prior to mass analysis. Spectral complexity

(Figure 7) only allowed the attribution of peaks corresponding to singly and doubly charged species (Table 4). The others peaks were poorly resolved, principally in the m/z 1400-2400 region: no significant deconvolution could hence be performed. Furthermore, taking into account possible spectral suppression effects, only 30% of the amino acid sequence could be recovered. Four Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

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Table 2. List of Peptides Arising from an In-Capillary 4-h Digestion of Hemoglobin origin

peaka

peptide

sequence

Mmeas (Mcalc), Da

mass spectrum (monoisotopic masses)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

β122-125 or β1 10-113 R30-33 R137-141 β81-87 R124-129 β103-109 R129-136 β18-26 or β102-109 R66-73 or R99-106 β32-40 β31-40 R111-123 β1-13 R107-141 β110-145 R1-106

(L)VVVL or (F)TPVL (L)ERMF (L)TSKYR (L)KGTFAAL (A)SLDKFL (F)KLLGNVL (F)LANVSTVL (V)KVDEVGGEA or (N)FKLLGNVL (A)LTKAVEHL or (F)KLLSHSLL (L)VVYPWTQRF (L)LVVYPWTQRF (A)SHLPSDFTPAVHA ()MLTAEEKAAVTAF (L)VTLASHLPSDFTPAVHASLDKFLANVSTVLTS (L)VVVLARNFGKEFTPVLQADFQKVVAGVANALA b

428.3 (428.3) 581.3 (581.3) 653.3 (653.4) 706.4 (706.4) 721.4 (721.4) 755.5 (755.5) 815.5 (815.5) 902.6 (902.6) 909.6 (909.6) 1194.7 (1164.6) 1307.8 (1307.7) 1377.7 (1377.7) 1380.7 (1380.7) 3787.0 (3787.2) 3966.3 (3966.5) 11283.8 (11284.2)

deconvoluted spectrum (average masses)

a The peak numbers indicated in this table are reproduced in the mass spectrum shown in Figure 5. b Sequence too long to be written in the table.

Figure 6. (a) Mass spectrum of a 0.5% acetic acid solution of reduced lysozyme, (b) corresponding deconvolution, and (c) mass spectrum of a 0.5% acetic acid solution of reduced lysozyme exposed to in-capillary pepsin for 1 h. Peak numbers and charge-state values correspond to the peptides listed in Table 3.

identified signals out of 30 were attributed to peptides obtained by nonspecific cleavages, 8 of them having several possible origins. The aforementioned examples clearly demonstrate the ease and reliability of this on-line protein digestion technique. A shorttime in-capillary exposure, depending on the structure order of the protein, ensures the enzyme site specificity and allows an 5428

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efficient digestion in term of quality of information provided by the mass spectrum. Moreover, the attribution of monoisotopic masses is significantly enhanced by using low cone voltage conditions. The mass spectrometric data, from a set of diagnostic ions, can be used not only for covering a whole sequence but also for protein identification from a database by means of an appropriate search software. The following section presents the feasibility of such an application using the peptide masses of each digested model protein. Protein Identification in Databases Using MS-Fit Software. As the software accepts either monoisotopic or exclusively average masses, the former was chosen for reasons of measurement accuracy and quantity of information. After selecting the database (e.g., Swiss Prot 10.05.99) and the enzyme used (pepsin from porcine gastric mucosa) and providing further information regarding possible modifications of amino acid residues such as cysteine or N- and C-terminal peptides, the investigation can be targeted to a certain kind of living species (e.g., mammals) and to molecular weight or isoelectric point (pI) ranges. The mass tolerance as well as the maximum number of missed cleavages and the minimum number of peptides required for matching must be indicated. In the case of the horse heart myoglobin digestion, the research was carried out by using the peptide masses presented in Table 1. The search was only limited to the molecular weight range 5000-20 000. The estimation of such a parameter can easily be obtained with an SDS-PAGE experiment or by a previous mass analysis. Species and pI windows remained unfulfilled. The mass tolerance was set at 0.3 Da. The minimum number of peptides required to match was set at seven. The maximum number of missed cleavages was set at 10. Search results show in the first four ranks myoglobins from different species, the first one being identified as horse myoglobin with 90% of identified peptides whereas the three others obtain only a score of 70%. Specifying the mammalian origin of the protein allowed an unambiguous answer. The attribution covered the complete peptide list, except the m/z value of 1708.7 originating from an unexpected cleavage.

Table 3. List of Peptides Issued from an In-Capillary Digestion of Reduced Lysozyme (1 h) origin

peaka

peptide

sequence

Mmeas (Mcalc), Da

mass spectrum (monoisotopic masses)

1 2 3 4 5 6 7 8 9 10 11

57-62 32-38 123-129 76-84 1-8 9-20 108-122 39-56 63-83 84-107 84-108

(L)QINSRW (A)AKFESNF (A)WIRGCRL (L)CNIPCSAL ()KVFGRCEL (L)AAAMKRHGLDNY (A)WVAWRNRCKGTDVQA (F)NTQATNRNTDGSTDYGIL (W)WCNDGRTPGSRNLCNIPCSAL (L)SSDITASVNCAKKIVSDGNGMNA (L)SSDITASVNCAKKIVSDGNGMNAW

802.6 (802.4) 841.6 (841.4) 902.7 (902.5) 932.6 (932.5) 950.7 (950.5) 1345.9 (1345.7) 1789.1 (1788.9) 1940.1 (1939.9) 2278.4 (2278.6) 2396.6 (2396.7) 2582.7 (2582.9)

deconvoluted spectrum (average masses) a

The peak numbers indicated in this table are reproduced in the mass spectrum shown in Figure 6c.

Figure 7. Mass spectrum of a 0.5% acetic acid solution of reduced albumin exposed to in-capillary pepsin for 1 h. Peak numbers and chargestate values correspond to the peptides listed in Table 4.

The 13 monoisotopic peptide masses used for the hemoglobin identification are taken from the upper part of Table 2. It was necessary to limit the molecular weight and pI ranges to 13 00018 000 and 6.5-8.5, respectively, to identify the multimer unambiguously. The case of hemoglobin, and more generally heteromultimers, is quite difficult to handle as the covering percentage of peptides attributed to a monomer is inevitably low. Nevertheless, a two-dimensional gel electrophoresis can be carried out prior to digestion so as to enter enough information in the parameters section, i.e., molecular weight and pI approximated values. The peptide masses corresponding to the digestion of reduced lysozyme used for the characterization are from the upper part of Table 3. Specifying the species Gallus gallus and a molecular weight range comprised between 5000 and 20 000 provided two answers, one of which being the C-precursor of lysozyme with seven peptides identified out of eight entered. With regard to lysozyme, the sequence of the C-precursor comprises 18 additional residues, corresponding to the signal peptide. The m/z value of 951.7, corresponding to the N-terminal octapeptide of lysozyme, was not recognized by the software, as the preceding residue in the sequence order of the C-precursor was not a convenient target

for pepsin. By changing the database to NCBInr 03.1.99, but without modifying any other parameter, four proteins were found. Among them, lysozyme was identified following 100% recognition of peptide data. This example reveals the lack of exhaustiveness of databases and the need to cross information in order to improve the degree of reliability that can be attributed to database search results. The 22 monoisotopic peptide masses measured from the reduced albumin digestion mixture were used for the database search (Table 4). Setting the species parameter to Bos taurus, having the molecular weight range between 50 000 and 70 000, and searching in four different databases gave a variety of results. However, in all cases, the precursor of bovine serum albumin was found (molecular weight of 69 294), differing from albumin by an N-terminal signal peptide containing 24 residues. NCBI was the only database from which albumin could be found as a fitting answer, placed at the third position but with many uncertainties concerning the sequence (some amino acids are reported as unknown residues). These investigations show that the combination of information, which issued from the homemade on-line digestion technique with Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

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Table 4. List of Peptides Issued from an In-Capillary 1-h Digestion of Reduced Albumin peaka

peptide

sequence

Mmeas (Mcalc), Da

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

346-348 75-80 455-459 569-574 or 20-25 228-233 or 523-528 349-354 568-574 or 20-26 213-218 b 20-27 568-576 219-227 250-259 or 491-500 70-80 377-386 c 27-36 or 223-233 346-356 d 215-226 355-373 182-205

(L)LRL (L)CKVASL (L)ILNRL (A)VEGPKL or (F)KGLVLI (F)VEVTKL or (I)KKQTAL (L)AKEYEA (F)AVEGPKL or (F)KGLVLIA (A)WSVARL b (F)KGLVLIAF (F)AVEGPKLVV (L)SQKFPKAEF (L)LECADDRADL or (L)TPDETYVPKA (L)FGDELCKVASL (L)KHLVDEPQNL c (A)FSQYLQQCPF or (F)PKAEFVEVTKL (L)LRLAKEYEATL d (S)VARLSQKPKAE (A)TLEECCAKDDPHACYSTVF (I)ETMREKVLASSARQRLRCASIQKF

400.3 (400.3) 619.5 (619.3) 627.5 (627.4) 641.5 (641.4;641.5) 687.5 (687.4;687.4) 709.5 (709.3) 712.5 (712.4;712.5) 730.6 (730.4) 749.5 (1) 859.6 (859.6) 910.7 (910.6) 1080.7 (1080.6) 1119.7 (1119.5;1119.6) 1180.8 (1180.6) 1191.8 (1191.6) 1202.9 (2) 1259.8 (1259.6;1259.7) 1305.7 (1305.7) 1350.9 (3) 1372.9 (1372.8) 2130.7 (2130.9) 2807.3 (2807.5)

a The peak numbers indicated in this table are reproduced in the mass spectrum shown in Figure 7. b Four peptides arising from aspecific cleavages can be attributed to the measured mass of 749.5 Da. c Six peptides corresponding to aspecific cleavages could correspond to the mass of 1202.9 Da. d Five peptides possessing a mass of 1350.9 Da could correspond to specific cleavages.

sequence database search, can allow identification of the injected protein, provided that some minimal information, concerning the molecular weight range, the pI range, or the kind of species that it comes from, is known. CONCLUSION The present work describes the making of a PEEK tube loaded and coated with nonsoluble pepsin and the use of it for protein digestion and identification. Experimental conditions could be optimized to obtain reliable and reproducible mass spectrometric results on the sequence data of several proteins of molecular weights between 14 000 and 66 000, with or without disulfide bridges. The complementary use of the MS-Fit software allows application of this technique to the identification of unknown proteins and, hence, should be of great interest in proteomic research. Furthermore, this on-line device is striking in the speed and ease of its setting up. By comparison with other on-line digestion procedures, the use of extemporaneous pepsin digestion offers a great versatility. It is thus possible to proceed successively to the mass measurement of an intact protein, pepsin precipitation, digestion, peptide analysis, and line regeneration in a single sequence. As a matter of fact, the duration of the whole experiment is only limited by the time of digestion. Additionally, this procedure does not necessitate any derivatization of the solid support, the protease, or the studied protein. In particular, a simple addition

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of dithiothreitol to the sample solution is sufficient to obtain an efficient digestion without any chemical protection step. It is also noteworthy that the length of PEEK tube defines the volume in which the digestion occurs. By simply cutting the line at the desired length, the reaction volume can be adapted to the available amount of sample (in the present study, a 1-m-long PEEK tube corresponded to 26 µL of protein solution). Toward this end, we are now optimizing this technique to the pico- and subpicomolar range by adapting the device to a microspray ion source. Low injection flows should allow multiple experiments such as MS/MS. Apart from the peptide masses deduced from the mass spectra, the sequence data obtained from MS/MS fragment ions are expected to provide a complementary way for protein identification from protein databases. ACKNOWLEDGMENT The authors gratefully acknowledge Dr. Peter Baker for helpful discussions and Dr. Bhupesh C. Das and Dr. Pierre Nahon for their assistance. We also thank the Association pour la Recherche contre le Cancer, France, for financial support. G.M. is indebted to the Ministe`re de l’Education Nationale, de la Recherche et de la Technologie, France for a Ph.D. research fellowship. Received for review April 11, 2000. Accepted August 22, 2000. AC000421Z