Direct quatitative analysis of native human peptides in complex secretome samples with PEAKS — ASN Events
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  3. Direct quatitative analysis of native human peptides in complex secretome samples with PEAKS

Direct quatitative analysis of native human peptides in complex secretome samples with PEAKS (#244)

Cassandra Wigmore 1 , Martjin Pinkse 2 , Bharath Kumar Raghuraman 2 , Baozhen Shan 1 , Peter D Verhaert 2
  1. BSI, Waterloo, Ontario, Canada
  2. Delft University of Technology, , Delft, Netherlands

Introduction

Native (poly)peptides released into body fluids contain highly relevant information as regulatory biomolecules with both diagnostic  and therapeutic potential. A major analytical challenge in secretory peptide researches is the low abundance of the analyte proportional to the large volume of extracellular matrix proteins. The current analytical solution to this problem is to enrich the peptide fraction of a complex sample by physical removal of the most abundant proteins. Here we report a direct approach to quantitative analysis of endogenous peptides in complex secretomes, without removal of highly abundant background proteins.

Methods

A high-resolution mass spectrometric workflow combining database search with de novo sequencing was proposed to facilitate analysis of the secretome beyond the dominant tryptic fragments. After a first round of database searching, with the tryptic enzyme specificity, all confident matches were filtered out. The remaining unidentified MS/MS spectra with good de novo sequencing tags were performed second round database search without enzyme specificity. This allows the identification of endogenous peptides with high sensitivity and accuracy. Peptides were quantified with intensity-based label-free quantification.

Results

Samples were conditioned FCS-containing medium of a selected population of human T-cells, which were reduced, alkylated, fractionated over C4 RP-HPLC, digested with trypsin and run by LC-MS with an LTQ Orbitrap Velos. Given a complex two-dimensional LC-MS/MS dataset (>130 GB) of conditioned medium of two differently treated primary human cell cultures sampled at three time points, PEAKS was supplemented with  the described novel data analysis workflow. This allows the identification of small endogenous peptides in the presence of a very busy background of predominant tryptic peptides, which made the former invisible in standard analyses. Label-free quantitation shows that these (poly)peptide profiles contain biomarkers for specific physiological or pathological conditions.

Conclusions
Direct analysis of endogenous peptides in complex secretomes