Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant.
Protein identification by peptide mass mapping usually involves digestion of gel-separated proteins with trypsin, followed by mass measurement of the resulting peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Positive identification requires measurement of enough peptide masses to obtain a definitive match with sequence information recorded in protein or DNA sequence databases. However, competitive binding and ionization of residual surfactant introduced during polyacrylamide gel electrophoresis (PAGE) can inhibit solid-phase extraction and MS analysis of tryptic peptides. We have evaluated a novel, acid-labile surfactant (ALS) as an alternative to sodium dodecylsulfate (SDS) for two-dimensional (2-D) PAGE separation and MALDI-MS mapping of proteins. ALS was substituted for SDS at the same concentration in buffers and gels used for 2-D PAGE. Manual and automated procedures for spot cutting and in-gel digestion were used to process Coomassie stained proteins for MS analysis. Results indicate that substituting ALS for SDS during PAGE can significantly increase the number of peptides detected by MALDI-MS, especially for proteins of relatively low abundance. This effect is attributed to decomposition of ALS under acidic conditions during gel staining, destaining, peptide extraction and MS sample preparation. Automated excision and digestion procedures reduce contamination by keratin and other impurities, further enhancing MS identification of gel separated proteins.
National Research Council of Canada, Plant Biotechnology Institute, Saskatoon SK, Canada. andrew.r.ross@ncr.ca
Andrew R S
AR
Ross
Peter J
PJ
Lee
Duncan L
DL
Smith
James I
JI
Langridge
Anthony D
AD
Whetton
Simon J
SJ
Gaskell
D000476:Alkanesulfonates / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D004148:Dioxolanes / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D010449:Peptide Mapping; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013501:Surface-Active Agents / Q000737:chemistry*
D000476:Alkanesulfonates; D004148:Dioxolanes; D011506:Proteins; D013501:Surface-Active Agents; C472187:sodium 4-((2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxyl)-1-propanesulfonate