Endogenous Plasma Peptide Detection and Identification in the Rat by a Combination of Fractionation Methods and Mass Spectrometry

Abstract

Mass spectrometry-based analyses are essential tools in the field of biomarker research. However, detection and characterization of plasma low abundance and/or low molecular weight peptides is challenged by the presence of highly abundant proteins, salts and lipids. Numerous strategies have already been tested to reduce the complexity of plasma samples. The aim of this study was to enrich the low molecular weight fraction of rat plasma. To this end, we developed and compared simple protocols based on membrane filtration, solid phase extraction, and a combination of both. As assessed by UV absorbance, an albumin depletion >99% was obtained. The multistep fractionation strategy (including reverse phase HPLC) allowed detection, in a reproducible manner (CV < 30%–35%), of more than 450 peaks below 3000 Da by MALDI-TOF/MS. A MALDI-TOF/MS-determined LOD as low as 1 fmol/μL was obtained, thus allowing nanoLC-Chip/MS/MS identification of spiked peptides representing ~10−6% of total proteins, by weight. Signal peptide recovery ranged between 5%–100% according to the spiked peptide considered. Tens of peptide sequence tags from endogenous plasma peptides were also obtained and high confidence identifications of low abundance fibrinopeptide A and B are reported here to show the efficiency of the protocol. It is concluded that the fractionation protocol presented would be of particular interest for future differential (high throughput) analyses of the plasma low molecular weight fraction.

Betaine Supplementation Causes an Increase in Fatty Acid Oxidation and Carbohydrate Metabolism in Livers of Mice Fed a High-Fat Diet: A Proteomic Analysis

“…Separation of peptides was performed on an analytical column. The eluted peptides were analyzed using a Q-Exactive mass spectrometer in the positive ion mode and data-dependent mode [ 19 ]. The MS data were obtained in full scan mode, with resolution power of 70,000 at 400 m / z .…”

Peptidomics Coming of Age: A Review of Contributions from a Bioinformatics Angle

“…78 Sequencing errors, mutations and homology searching can sometimes be taken into account. Looking into recent peptidomics projects it is notable that manual de novo sequencing is still frequently performed, [79][80][81] sometimes combined with automated de novo tools like Peaks, [81][82][83][84] MS-Tag, 85 or PepSeq (Micromass Co., Manchester, U.K.). 80 Other tools, aiming for novel peptide discovery, scan the complete genome translated in its six reading frames.…”