Protein identification by syringe pump-driven reversed-phase LC-MS/MS.
In typical mass spectrometry-based protein identification using peptide fragmentation fingerprinting, front-end separation plays a critical role in successful peptide sequencing. This separation step demands a great deal of time and usually is the rate-limiting step for the whole process. Here we provide an alternative separation method, based on a simple nanoflow delivery system, that is able to shorten the separation time considerably. This system consists of a 25-mul syringe connected to a manually packed reversed-phase mini-capillary column that can be directly coupled to an electrospray ionization tandem mass spectrometer. A syringe pump is then used to deliver the peptide mixtures at a nanoscale flow rate. We examined the efficiency and efficacy of this method by analyzing the tryptic peptides of bovine serum albumin and of 10 Escherichia coli proteins separated by two-dimensional gel electrophoresis (2DE). The results showed that identification of each protein could be achieved successfully within 25 min by using the disposable mini-capillary column. Moreover, all 2DE-separated E. coli proteins were identified at high confidence levels. Together, our data suggest that this method is a suitable option for mass spectrometry-based protein identification.
Lu
Chi-Yu
CY
Proteome Research Center, National Yang-Ming University, Taipei 11221, Taiwan.
Wu
Chiu-Yi
CY
Lin
Chao-Hsiung
CH
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000295:instrumentation* / Q000379:methods*; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012394:Rosaniline Dyes; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization; D013594:Syringes; D053719:Tandem Mass Spectrometry
D010455:Peptides; D011506:Proteins; D012394:Rosaniline Dyes; D012997:Solvents; D012710:Serum Albumin, Bovine; C048139:Coomassie blue