Rapid identification of comigrating gel-isolated proteins by ion trap-mass spectrometry.
作者： David Arnott , William J. Henzel , John T. Stults
关键词: Protein mass spectrometry 、 Tandem mass spectrometry 、 Mass spectrometry 、 Bottom-up proteomics 、 Selected reaction monitoring 、 Chromatography 、 Top-down proteomics 、 Chemistry 、 Analytical chemistry 、 Peptide mass fingerprinting 、 Peptide sequence tag
摘要: In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were analyzed and each was found to contain number of proteins that subsequently identified by tandem mass spectrometry (MS/MS) on quadrupole ion trap instrument. The digested with trypsin in situ polyvinylidene difluoride (PVDF) membrane following electroblot transfer. Analysis 2.5% aliquot peptide mixture matrix assisted laser desorption/ionization-mass (MALDI-MS) followed an initial database masses failed identify proteins. peptides separated reversed-phase capillary high performance liquid chromatography (HPLC) anticipation subsequent Edman degradation, but analysis chromatographic fractions MALDI-MS revealed multiple, coeluting precluded this approach. Selected HPLC-electrospray ionization-ion spectrometry. Tandem provided significant fragmentation which full or partial sequence deduced peptides. Two stages (MS3) used one case determine additional sequence. Database searches, using single plus sequence, four electrophoretic band at 45 kDa, second 60 kDa. Many these derived human keratin. protein identifications corroborated presence multiple matching spectra. addition, not DNA databases, determined interpretation MS/MS data. These results demonstrate power identification mixture, de novo determination Reanalysis data modified searching algorithm showed same sets limited fragment masses, absence spectral amino acid implications solely are discussed, including advantages low signal levels, reduction necessary expertise, increased speed.
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