Issues and Applications in Label-Free Quantitative Mass Spectrometry

Department of Cellular & Integrative Physiology, Biotechnology Research & Training Center, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
International journal of proteomics 01/2013; 2013(1):756039. DOI: 10.1155/2013/756039
Source: PubMed

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.

Download full-text


Available from: Lianshui Wang, Aug 12, 2014
18 Reads
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 hexposure to 10 μg/mL and 100 ng/mL of two common carbon nanoparticles, single- and multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study the differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformaticanalysis of proteins identified in LFQMS. Interestingly, after exposure to ahigh concentration (10 μg/mL; 0.4 μg/cm(2)) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm(2)) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins in common to both nanotubes occurred within the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The majority of the protein changes represent a decrease in amount suggesting a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure.
    10/2013; 1(3):219-239. DOI:10.3390/proteomes1030219
  • [Show abstract] [Hide abstract]
    ABSTRACT: Difference gel electrophoresis enables the accurate quantification of changes in the proteome including combinations of post-translational modifications and protein isoform expression. Here we review recent advances in study design, image acquisition and statistical analysis. We also compare DIGE to established and emerging mass spectrometric analysis technologies. Despite these recent advances in mass spectrometry and the still unsolved limitations of 2DE to map hydrophobic, high molecular weight and proteins with extreme pIs, DIGE remains the most comprehensive top-down method to study changes in abundance of intact proteins.This article is protected by copyright. All rights reserved
    PROTEOMICS - CLINICAL APPLICATIONS 11/2014; 9(3-4). DOI:10.1002/prca.201400119 · 2.96 Impact Factor