Isocratic solid phase extraction-liquid chromatography (SPE-LC) interfaced to high-performance tandem mass spectrometry for rapid protein identification.
Reversed-phase liquid chromatography interfaced to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) allows analysis of very complex peptide mixtures at great sensitivity, but it can be very time-consuming, typically using 60 min, or more, per sample analysis. We recently introduced the isocratic solid phase extraction-liquid chromatography (SPE-LC) technology for rapid separation (~8 min) of simple peptide samples. We now extend these studies to demonstrate the potential of SPE-LC separation in combination with a hybrid linear ion trap-Orbitrap tandem mass spectrometer for efficient analysis of peptide samples in proteomics research. The system performance of SPE-LC-MS/MS was evaluated in terms of sensitivity and efficiency for the analysis of tryptic peptide digests obtained from samples consisting of up to 12 standard proteins. The practical utility of the analytical setup was demonstrated by the analysis of <15 microg depleted human serum proteome by a combination of SDS-PAGE and SPE-LC-MS/MS. A total of 88 unique gene products spanning 3 orders of magnitude in serum protein concentration were identified using stringent database search criteria.
Hørning
Ole B
OB
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, Denmark.
Kjeldsen
Frank
F
Theodorsen
Søren
S
Vorm
Ole
O
Jensen
Ole N
ON
D000818:Animals; D001798:Blood Proteins / Q000032:analysis; D002417:Cattle; D002645:Chickens; D002853:Chromatography, Liquid / Q000295:instrumentation; D016208:Databases, Factual; D006801:Humans; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D040901:Proteomics; D012680:Sensitivity and Specificity; D044967:Serum; D052616:Solid Phase Extraction / Q000295:instrumentation; D053719:Tandem Mass Spectrometry
D001798:Blood Proteins; D010455:Peptides; D011506:Proteins