An assessment of peptide enrichment methods employing mTRAQ quantification approaches.
AffiliationSchool of Cancer & Enabling Sciences, Wolfson Molecular Imaging Centre, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK.
MetadataShow full item record
AbstractThe human plasma peptidome has potential in biomarker discovery not least because the plasma proteome is a challenging matrix due to its complexity and dynamic range. However, methods to significantly reduce the amount of protein present in plasma while retaining the less abundant peptides present in plasma samples has been a major issue. Here, we present a novel strategy which has been employed to assess the effectiveness of removing interfering proteins while retaining peptides of interest. To monitor peptide retention, a spiked in digested protein, in this case a synthetic QconCAT protein, was employed. This enabled a variety of target analytes (peptides) to be monitored for their retention in liquid phase, providing a broader picture of peptide loss from each method assessed. The incorporation of mTRAQ labeling allowed the presence of each peptide to be monitored, and accurate peptide losses to be determined in a Selected Reaction Monitoring (SRM) assay, thus, enabling an objective semiquantitative conclusion to be drawn regarding the suitability of each method for protein removal and peptide retention. We also assessed a range of methods for retaining nontryptic peptides in a plasma peptidomics workflow. From these data, we determined an optimal workflow for removing intact protein, while retaining peptides for MS-based analyses.
CitationAn assessment of peptide enrichment methods employing mTRAQ quantification approaches. 2012, 84 (13):5604-10 Anal Chem
- Reversed-phase high-performance liquid chromatographic prefractionation of immunodepleted human serum proteins to enhance mass spectrometry identification of lower-abundant proteins.
- Authors: Martosella J, Zolotarjova N, Liu H, Nicol G, Boyes BE
- Issue date: 2005 Sep-Oct
- Unmasking low-abundance peptides from human blood plasma and serum samples by a simple and robust two-step precipitation/immunoaffinity enrichment method.
- Authors: Halfinger B, Sarg B, Amann A, Hammerer-Lercher A, Lindner HH
- Issue date: 2011 Jun
- Evaluation of endogenous plasma peptide extraction methods for mass spectrometric biomarker discovery.
- Authors: Aristoteli LP, Molloy MP, Baker MS
- Issue date: 2007 Feb
- Peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display.
- Authors: Tammen H, Schulte I, Hess R, Menzel C, Kellmann M, Mohring T, Schulz-Knappe P
- Issue date: 2005 Aug
- Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis.
- Authors: Kim JY, Lee JH, Park GW, Cho K, Kwon KH, Park YM, Cho SY, Paik YK, Yoo JS
- Issue date: 2005 Aug