Issues and applications in label-free quantitative mass spectrometry.

Xianyin Lai,Lianshui Wang,Frank A Witzmann

doi:10.1155/2013/756039

Abstract

To address the challenges associated with differential expression proteomics, label-free mass spectrometric protein quantification methods have been developed as alternatives to array-based, gel-based, and stable isotope tag or label-based approaches. In this paper, we focus on the issues associated with label-free methods that rely on quantitation based on peptide ion peak area measurement. These issues include chromatographic alignment, peptide qualification for quantitation, and normalization. In addressing these issues, we present various approaches, assembled in a recently developed label-free quantitative mass spectrometry platform, that overcome these difficulties and enable comprehensive, accurate, and reproducible protein quantitation in highly complex protein mixtures from experiments with many sample groups. As examples of the utility of this approach, we present a variety of cases where the platform was applied successfully to assess differential protein expression or abundance in body fluids, in vitro nanotoxicology models, tissue proteomics in genetic knock-in mice, and cell membrane proteomics.

Highlights

Protein quanti cation for differential expression analysis or expression pro ling represents the most challenging aspect in proteomics technology. is task is typically carried out through array-based [1], two-dimensionalelectrophoretic (2-DE-) based [2] or mass-spectrometry(MS-) based approaches [3, 4]
In bottom-up quantitative approaches, complex protein mixtures are digested enzymatically, peptides from each protein are separated by liquid chromatography (LC) and detected by MS, and protein quanti cation is completed at the peptide level and combined to calculate a summarized value for the protein from which they come
Peak area has been applied extensively in the quanti cation of small molecule compounds [12,13,14] and is the most reliable measurement for quanti cation. ecause peptides perform to small molecule compounds in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, direct measurement of peak area is considered a reliable method for peptide and protein quanti cation. umerous so ware packages for label-free quanti cation of LCMS/MS-derived data based on peptide peak area have been developed and applied in proteomics research

Summary

Introduction

Protein quanti cation for differential expression analysis or expression pro ling represents the most challenging aspect in proteomics technology. is task is typically carried out through array-based [1], two-dimensionalelectrophoretic (2-DE-) based [2] or mass-spectrometry(MS-) based approaches [3, 4]. Abundance, such as the peptide peak intensity (height or area of a peak), the peptide precursor ion peak height, and the peak height of product ions, can be extracted Using such information individually or combinatorially, numerous label-free methods have been developed, including two extensively applied but fundamentally different strategies: quantitation based on spectral counting [10] and peptide ion peak area [11]. Data acquisition in this manner is most typical when the dynamic exclusion mode is applied to identify substantially more peptides. Ecause peptides perform to small molecule compounds in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, direct measurement of peak area is considered a reliable method for peptide and protein quanti cation.

Issues

Findings

Conclusion