ISSN:
0173-0835
Keywords:
Mass spectrometry
;
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
;
Electrospray ionization ion-trap mass spectrometry
;
In-gel proteolysis
;
Proteome analysis
;
Capillary high performance liquid chromatography
;
Two-dimensional polyacrylamide gel electrophoresis
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Two-dimensional polyacrylamide gel electrophoresis (2-DE) in combination with mass spectrometry is an extremely powerful tool for characterizing complex mixtures of proteins. In many cases, the success of this approach relies upon the ability to recover peptides at high concentrations and free of interfering artifacts from in-gel and/or on-membrane enzymatic digests. In previous studies, we demonstrated that capillary or microcolumn ( ≤ 350 μm ID) rever-sed-phase high performance liquid chromatography (RP-HPLC) is a powerful microseparation technique for proteins and peptides (Moritz, R. L. and Simpson, R. J., J. Chromatogr. 1992, 599, 119-130). Here we evaluate various capillary column RP-HPLC/mass spectrometric approaches for identifying and characterizing 2-DE resolved proteins. For these studies, stable and efficient 0.20 mm and 0.32 mm internal diameter (ID) fused-silica columns with hydrophilic polyvinylidene difluoride (PVDF) frits were fabricated and slurry packed with 7 μm spherical, 300 Å pore size, C8 bonded phase silica particles. We show that capillary column chromatography is a rapid and efficient desalting/concentrating (ON/OFF) technique for sample cleanup prior to protein identification by peptide-mass fingerprinting using matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry. While marginally more peptide mass information can be obtained by stepped elution of the peptide mixture with increasing concentrations of organic solvent, best results were obtained by fractionation of the peptide mixture using a linear 60 min gradient. One salient feature of this study was the observation that, in contrast to the stepped elution and gradient approaches, the ionization of peptide T1 (m/z 2402.2 SGETEDTFIADLVV(PeCys)TGQIK) was almost completely suppressed using the ON/OFF approach. Maximal amino acid sequence coverage, a necessary prerequisite for complete characterization of a protein, was accomplished using a capillary column (0.2 mm ID) directly coupled with an electro-spray ionization (ESI) ion-trap tandem mass spectrometer. For example, from an in situ tryptic digest of α-enolase isolated by 2-DE from the human breast carcinoma cell line MDA-MB231, 71% of the amino acid sequence was obtained. In addition to identifying two possible N-terminal acetylated α-enolase variants, Asn153Asp and Ile152Asp/Asn153Ile, the tandem mass spectrometric data revealed the presence of a number of process-induced modifications of α-enolase such as methionine oxidation and cysteine amidoe-thylationPart of this work was presented at ICES '97, International Council of Electrophoretic Societies Symposium, held March 23-27, 1997 in Seattle, WA, USA.
Additional Material:
10 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150190610
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