Automated analysis of mouse serum peptidome using restricted access media and nanoliquid chromatography–tandem mass spectrometry

Abstract

We demonstrate use of restricted access media with reversed phase functionality (RAM-RP) for analysis of low molecular weight proteins and peptides in mouse serum (75 μl) using a custom designed modular automated processing system (MAPS). RAM-RP fractionation with simultaneous removal of high molecular weight and high abundance proteins is integrated with a follow-on buffer exchange module (BE) to ensure compatibility with subsequent processing steps (trypsin digestion and intact peptide separation prior to mass spectrometric analysis). The high sample capacity afforded by chromatographic methods generates enough sample to achieve comprehensive serum peptidome identification (357 proteins) through tandem mass spectrometric analysis of both intact and digested peptides. Sample losses during transfer between modules are minimized through precise fluidic control; no clogging occurred over several months of serum processing in our low back pressure system. Computer controlled operation of both modules and thorough optimization yield excellent run-to-run reproducibility and protein/peptide overlap in analytical repeats. The robustness of our results demonstrate that the RAM-RP-BE workflow executed on our MAPS platform shows tremendous potential for high throughput peptidome processing, particularly with regard to direct analysis of small-volume serum samples.

Can Proteomic Profiling Identify Biomarkers and/or Therapeutic Targets for Liver Fibrosis?

“…Finally, peptide mass is computed from the time spend to reach the detector ("time-of-flight"). Another frequently applied method is LC-MS/MS which efficiently profiles large numbers of samples with the analysis of extremely small volume samples (i.e., <75 µl) by evaluating proteins with masses ranging from 2 to 200 kDa with tremendous efficiency and reasonable reproducibility [65]. In addition, SELDI-TOF MS, which couples a prefractination stage with MALDI-TOF, is currently used for proteomic profiling studies.…”

Introduction

“…Comprehensive analysis of serum peptidome using restricted access media and nanoliquid chromatographyetandem mass spectroscopy (Gil et al, 2010). It is also the fourth leading cause of death in men, and the fifth leading cause of death in women.…”

MicroQuEChERS–nanoliquid chromatography–nanospray–tandem mass spectrometry for the detection and quantification of trace pharmaceuticals in benthic invertebrates

“…For example, using an electrospray ionisation interface as a continuous flow ionisation technique enhances the ionisation process because the number of ions increases in the gas phase, thereby improving the sensitivity [43]. The largest application for nano-LC is proteomic analysis [44][45][46]; however, it has not been as widely applied in other important fields such as environmental analysis. Consequently, it is necessary to explore the potential of nano-LC in environmental field analysis.…”