A novel interface for variable flow nanoscale LC/MS/MS for improved proteome coverage.
A variable flow "peak trapping" liquid chromatography (LC) interface has been developed for the coupling of nanoscale LC to electrospray ionization mass spectrometry (ESI-MS). The presented peak trapping LC interface allows for the extended analysis time of co-eluting compounds and has been employed for the identification of proteins via tandem mass spectrometry (MS/MS). The variable flow process can be controlled either manually or in a completely automated manner where the mass spectrometer status determines the status of the variable flow interface. When the mass spectrometer operates in MS survey mode, the interface is operated in a so-called "high-flow" mode. Alternatively, the interface is operated in a "low-flow" mode during MS/MS analysis. In the "high-flow" mode of the variable flow process the column flow rate is typically around 200 nL/min, whereas in the "low-flow" mode the column effluent is introduced into the source of the mass spectrometer at 25 nL/min. In addition to the flow reduction during MS/MS analysis, the gradient is paused to preserve the peptide separation on the analytical nanoscale LC column. The performance of the variable flow nanoscale LC/MS/MS interface is demonstrated by the automated analysis of standard peptide mixtures and protein digests utilizing variable flow, data-dependent scanning MS/MS techniques, and automated database searching.
Vissers
Johannes P C
JP
LC Packings, Baarsjesweg, Amsterdam, The Netherlands.
Blackburn
R Kevin
RK
Moseley
M Arthur
MA
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D003627:Data Interpretation, Statistical; D007202:Indicators and Reagents; D008928:Mitochondria / Q000737:chemistry; D009496:Neurotensin / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D012339:RNA, Ribosomal, 28S / Q000737:chemistry; D012269:Ribosomal Proteins / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013954:Thyroglobulin / Q000737:chemistry
D007202:Indicators and Reagents; D020543:Proteome; D012339:RNA, Ribosomal, 28S; D012269:Ribosomal Proteins; D009496:Neurotensin; D013954:Thyroglobulin