Rapid mass spectrometric identification of proteins from two-dimensional polyacrylamide gels after in gel proteolytic digestion.
We report a rapid method for identifying proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). In-gel digestion was performed in a way such that the volume ratio of trypsin solution to gel plug was quantitatively controlled to promote reproducible digestion and to maximize the digestion yield. To make the digestion samples more compatible with MALDI-MS, the volatile salt ammonium bicarbonate in the digestion buffer was largely removed prior to peptide extraction. Samples of mixed tryptic peptides from in-gel digestion were used without purification to obtain molecular weights by MALDI-MS with alpha-cyano, 4-hydroxy-cinnamic acid as the matrix. Modifications of MALDI sample loading procedures improved the detection sensitivity by one half to one order of magnitude. The peptide mass peaks in MALDI-MS spectra were distinguished from those of impurities by using several types of controls, and masses were corrected by using trypsin autodigestion fragments as internal calibration standards. Two different peptide-matching computer programs were used to interrogate sequence databases and identify proteins. Identification was enhanced by generation of orthogonal data sets (by using different proteases) and by including experimental values of isoelectric point (pI) and molecular weight to exclude false entries in the candidate lists. Approximately 1% of the material from a spot was used in each sample loading, and nine protein spots from rat liver 2-D PAGE gels were identified correctly, as judged by comparison with identification results previously obtained from Edman sequencing. A previously identified low-abundance spot was not identified by MALDI-MS, presumably because there was insufficient material in a single gel. The sample handling procedure reported here should permit us to identify many 2-D PAGE protein spots of medium abundance.
Li
G
G
Laboratory of Molecular Pharmacology, National Cancer Institute (NCI), NIH, Bethesda, MD 20892-4255, USA.
Waltham
M
M
Anderson
N L
NL
Unsworth
E
E
Treston
A
A
Weinstein
J N
JN
D000180:Acrylic Resins; D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D005782:Gels; D008099:Liver / Q000737:chemistry; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis*; D051381:Rats; D011916:Rats, Inbred F344; D012697:Serine Endopeptidases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism*
D000180:Acrylic Resins; D005782:Gels; D011506:Proteins; C016680:polyacrylamide gels; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase