Online Nanoflow Reversed Phase-Strong Anion Exchange-Reversed Phase Liquid Chromatography―Tandem Mass Spectrometry Platform for Efficient and In-Depth Proteome Sequence Analysis of Complex Organisms
In: Analytical chemistry (Washington), Jg. 83 (2011), Heft 18, S. 6996-7005
academicJournal
- print, 71 ref
Zugriff:
The dynamic range of protein expression in complex organisms coupled with the stochastic nature of discovery-driven tandem mass spectrometry (MS/MS) analysis continues to impede comprehensive sequence analysis and often provides only limited information for low-abundance proteins. High-performance fractionation of proteins or peptides prior to mass spectrometry analysis can mitigate these effects, though achieving an optimal combination of automation, reproducibility, separation peak capacity, and sample yield remains a significant challenge. Here we demonstrate an automated nanoflow 3-D liquid chromatography (LC)—MS/MS platform based on high-pH reversed phase (RP), strong anion exchange (SAX), and low-pH reversed phase (RP) separation stages for analysis of complex proteomes. We observed that RP-SAX-RP outperformed RP-RP for analysis of tryptic peptides derived from Escherichia coli and enabled identification of proteins present at a level of 50 copies per cell in Saccharomyces cerevisiae, corresponding to an estimated detection limit of 500 amol, from 40 μg of total lysate on a low-resolution 3-D ion trap mass spectrometer. A similar study performed on a LTQ-Orbitrap yielded over 4000 unique proteins from 5 μg of total yeast lysate analyzed in a single, 101 fraction RP-SAX-RP LC—MS/MS acquisition, providing an estimated detection limit of 65 amol for proteins expressed at 50 copies per cell.
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Online Nanoflow Reversed Phase-Strong Anion Exchange-Reversed Phase Liquid Chromatography―Tandem Mass Spectrometry Platform for Efficient and In-Depth Proteome Sequence Analysis of Complex Organisms
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Autor/in / Beteiligte Person: | FENG, ZHOU ; SIKORSKI, Timothy W ; FICARRO, Scott B ; WEBBER, James T ; MARTO, Jarrod A |
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Zeitschrift: | Analytical chemistry (Washington), Jg. 83 (2011), Heft 18, S. 6996-7005 |
Veröffentlichung: | Washington, DC: American Chemical Society, 2011 |
Medientyp: | academicJournal |
Umfang: | print, 71 ref |
ISSN: | 0003-2700 (print) |
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