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 microm ID) reversed-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 microm spherical, 300 A 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 electrospray ionization (ESI) ion-trap tandem mass spectrometer. For example, from an in situ tryptic digest of
alpha-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
alpha-enolase variants, Asn153Asp and Ile152Asp/Asn153Ile, the tandem mass spectrometric data revealed the presence of a number of process-induced modifications of
alpha-enolase such as
methionine oxidation and
cysteine amidoethylation.