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Zhou F, et al.  (2011) 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. Anal Chem 83(18):6996-7005

Abstract: 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 ?g 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 ?g 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.

Status: Published Type: Journal Article PubMed ID: 21851055

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Topics Topics not linked to Genes Genes linked to topics (#1 - 10 )
CHL1 DSE4 HEF3 INP51 MIP6 MLH2 PMC1 RPT1 SDS3 SPO14
Additional Literature blue ball blue ball blue ball blue ball blue ball blue ball blue ball blue ball blue ball blue ball
Large-scale protein detection yg ball
Omics yg ball
Protein Physical Properties blue ball blue ball blue ball blue ball blue ball blue ball blue ball blue ball blue ball blue ball
Techniques and Reagents yg ball

Topics Genes linked to topics (#11 - 15 )
VPS13 YIL092W YKL075C YOR093C ZDS2
Additional Literature blue ball blue ball blue ball blue ball blue ball
Protein Physical Properties blue ball blue ball blue ball blue ball blue ball

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