Protein Fractionation for Quantitative Plasma Proteomics by Semi-Selective Precipitation

Ekaterina Mostovenko

doi:10.4172/jpb.1000239

Abstract

Blood plasma is a highly complex mixture of proteins, metabolites and lipids, and a rich source of potential biomarkers for a range of diseases and conditions. The wide range in protein abundance poses a tremendous challenge for plasma proteomics. However, as a relatively small number of proteins make up most of the total protein pool, the concentration range can be compressed by depletion of abundant proteins, such as albumin. To reduce sample complexity and increase the protein coverage, we have developed a sample preparation method based on semi-selective precipitation with acetonitrile at different pH and built a data analysis pipeline, combining different search strategies. The method we propose is reproducible and easily parallelised (high throughput), and may be well suited to fractionate plasma for label-free quantitative proteomics in large clinical studies. Up to 90% of albumin and other abundant proteins were removed by adding an equal volume of acetonitrile to the samples adjusted to pH 5.

Highlights

IntroductionLipids, salts, vitamins, amino acids, nucleic acids, hormones and around 75 mg/mL protein [1]
Plasma contains carbohydrates, lipids, salts, vitamins, amino acids, nucleic acids, hormones and around 75 mg/mL protein [1]
The protein concentration was defined using a Bicinchoninic Acid (BCA) protein assay kit (Thermo Fisher Scientific). This protein extraction reagent has been developed for the lysis and protein solubilisation from bacteria, but is routinely used in our laboratory and directly compatible with BCA analysis, SDS-PAGE, tryptic digestion, and samples are cleaned up for analysis by Liquid Chromatography-Mass Spectrometry (LC-MS)

Summary

Introduction

Lipids, salts, vitamins, amino acids, nucleic acids, hormones and around 75 mg/mL protein [1]. The carrier-protein albumin dominates with 45-50% of the total protein concentration, while immunoglobulin G and transferrin contribute 8-20% and 3-7%, respectively [2]. These and other highly abundant, large proteins mask less abundant ones by decreasing their relative concentration, and through effects such as ion suppression in electrospray ionization mass spectrometry. Changes in the abundant proteins may be indicative of the physiological status of the organism,[3] lowabundant proteins, for instance from tissue leakage, may mark an early state of a disease such as cancer [4,5]. Plasma is sampled, the concentration range of proteins, spanning from picogram to microgram per millilitre, is a major challenge in clinical proteomics

Methods

Results

Conclusion