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J Biomol Tech. 2007 February; 18(1): 12.
PMCID: PMC2291845

P32-M HT Proteomics LC/MS For Analysis of Low-Abundance Proteins, PTMs, and Biomarkers

Abstract

The introduction of electrospray ionization (ESI) in the 1980s revolutionized the analysis of biomolecules, and LC-ESI/MS (50–5000 μL/min) offers robust, high-throughput analyses for many biological applications. The introduction of nanospray (nESI) in the 1990s has made nLC-nESI/MS (10–1000 nL/min) a valuable tool for proteomics research, where high resolution and high sensitivity are maximized at the cost of sample throughput (60–240+ min per analysis) and robustness. Although protein biomarker analysis requires the high resolution and high sensitivity provided by nanoLC/MS, it also requires robustness and high throughput if it is to be useful in pharmaceutical and clinical applications. This study introduces a new axial desolvation, vacuum-assisted nano-capillary electrospray (ADVANCE) source for LC/MS, which has the robustness of LC-ESI/MS and the sensitivity of nanoLC-nanoESI/MS, but operates in the flow range from 0.2–100 μL/min. For low-abundance proteins, PTMs, and biomarker identification, an HT capLC-ADVANCE/MS system can analyze 50–150 host cell proteomic samples (digests of 1D gel slices, 2D gel spots, or MDLC fractions) in 24 h (10–30 min per analysis) at attomole to picomole sensitivities. The capLC-ADVANCE/MS system can also be used for validation of biomarker candidates, which requires comparative quantitation (ICAT, ITRAC, SILAC) of proteomic samples (5–15 min per analysis) in host cells and physiological fluids. Once protein biomarkers have been validated (very few to date), the capLC-ADVANCE/MS can be used for very high throughput (2–5 min per analysis) quantitation of biomarker signature peptides in a variety of diagnostic and therapeutic applications.


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