On-column digestion of proteins in aqueous-organic solvents

被引:95
作者
Slysz, GW [1 ]
Schriemer, DC [1 ]
机构
[1] Univ Calgary, Dept Mol Biol & Biochem, Calgary, AB T2N 4N1, Canada
关键词
D O I
10.1002/rcm.1022
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Proteolytic digestion is an important step in protein identification by peptide mass mapping and tandem mass spectrometry (MS/MS)-based peptide sequencing. Traditional methods of protein digestion require extended incubation times and have difficulty with proteolytically resistant proteins. Here, we describe a method in which a protein solution was combined with a mixed aqueous-organic solution (methanol, isopropanol, or acetonitrile) and passed through a microcolumn containing immobilized trypsin. Myoglobin sequence coverage was high (>85%) in all three solvents, and differences in spectra were seen among the different solution conditions. Notably, methanol-based digestions produced fewer missed cleavages while acetonitrile-based digestions produced the most peptides and the most intense mass spectra. Flow rates through the column were varied from 0.5 to 15 muL/min, corresponding to column residence times of 78 and 2.6 s, respectively. All flow rates produced high sequence coverage of myoglobin, although, at higher flow rates, more missed cleavages were observed. No significant increase in undigested myoglobin was observed with flow rates up to 15 muL/min. The described method was applied to the digestion of human transferrin (hTf), a proteolytically resistant protein. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis detected 42 peptides covering 46% of the hTf sequence. The traditional aqueous method resulted in 12 peptides (8% sequence coverage) only when high concentrations of trypsin were used. Lastly, digestion of low nanomolar myoglobin was shown to produce detectable peptides and resulted in a correct database hit. Thus, we demonstrate a method that is capable of rapid on-line digestion, thereby lending itself to high-throughput identification of proteins. Copyright (C) 2003 John Wiley Sons, Ltd.
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收藏
页码:1044 / 1050
页数:7
相关论文
共 32 条
[1]   Overalkylation of a protein digest with iodoacetamide [J].
Boja, ES ;
Fales, HM .
ANALYTICAL CHEMISTRY, 2001, 73 (15) :3576-3582
[2]   EFFECT OF TRYPSIN ON BOVINE TRANSFERRIN AND LACTOFERRIN [J].
BROCK, JH ;
ARZABE, F ;
LAMPREAVE, F ;
PINEIRO, A .
BIOCHIMICA ET BIOPHYSICA ACTA, 1976, 446 (01) :214-225
[3]   Confocal fluorescence microscopic imaging for investigating the analyte distribution in MALDI matrices [J].
Dai, YQ ;
Whittal, RM ;
Li, L .
ANALYTICAL CHEMISTRY, 1996, 68 (15) :2494-2500
[4]   RAPID TRYPTIC MAPPING USING ENZYMATICALLY ACTIVE MASS-SPECTROMETER PROBE TIPS [J].
DOGRUEL, D ;
WILLIAMS, P ;
NELSON, RW .
ANALYTICAL CHEMISTRY, 1995, 67 (23) :4343-4348
[5]   Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass measurement accuracy by using delayed extraction [J].
Edmondson, RD ;
Russell, DH .
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, 1996, 7 (10) :995-1001
[6]   Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification [J].
Gao, J ;
Xu, JD ;
Locascio, LE ;
Lee, CS .
ANALYTICAL CHEMISTRY, 2001, 73 (11) :2648-2655
[7]   Rapid micro-scale proteolysis of proteins for MALDI-MS peptide mapping using immobilized trypsin [J].
Gobom, J ;
Nordhoff, E ;
Ekman, R ;
Roepstorff, P .
INTERNATIONAL JOURNAL OF MASS SPECTROMETRY, 1997, 169 :153-163
[8]   On protein denaturation in aqueous-organic mixtures but not in pure organic solvents [J].
Griebenow, K ;
Klibanov, AM .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1996, 118 (47) :11695-11700
[9]   Advances in proteome analysis by mass spectrometry [J].
Griffin, TJ ;
Aebersold, R .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (49) :45497-45500
[10]  
Herbert B, 2001, ELECTROPHORESIS, V22, P2046, DOI 10.1002/1522-2683(200106)22:10<2046::AID-ELPS2046>3.0.CO