Signature-peptide approach to detecting proteins in complex mixtures.
The objective of the work presented in this paper was to test the concept that tryptic peptides may be used as analytical surrogates of the protein from which they were derived. Proteins in complex mixtures were digested with trypsin and classes of peptide fragments selected by affinity chromatography, lectin columns were used in this case. Affinity selected peptide mixtures were directly transferred to a high-resolution reversed-phase chromatography column and further resolved into fractions that were collected and subjected to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The presence of specific proteins was determined by identification of signature peptides in the mass spectra. Data are also presented that suggest proteins may be quantified as their signature peptides by using isotopically labeled internal standards. Isotope ratios of peptides were determined by MALDI mass spectrometry and used to determine the concentration of a peptide relative to that of the labeled internal standard. Peptides in tryptic digests were labeled by acetylation with acetyl N-hydroxysuccinimide while internal standard peptides were labeled with the trideuteroacetylated analogue. Advantages of this approach are that (i) it is easier to separate peptides than proteins, (ii) native structure of the protein does not have to be maintained during the analysis, (iii) structural variants do not interfere and (iv) putative proteins suggested from DNA databases can be recognized by using a signature peptide probe.
Geng
M
M
Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
Ji
J
J
Regnier
F E
FE
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
D010455:Peptides; D011506:Proteins; D014357:Trypsin