Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry

Abstract

Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of <12%. Using isobaric tags for relative and absolute quantitation (iTRAQ) 4-plex, >4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.

Figure 1

Overview of multiplexed workflow for discovery proteomics in plasma. This figure shows application of the workflow to a specific study in which iTRAQ 4-plex reagent was used to process and analyze four time points of planned myocardial infarction (PMI) patient samples. However, as shown in the protocol, other depletion and labeling strategies can be used to accommodate the goals of a given study. HCD, higher energy collisional dissociation. Image adapted with permission from Keshishian et al.38, American Society for Biochemistry and Molecular Biology.

Figure 2

Chromatogram of plasma after immunoaffinity depletion. (a,b) Plasma chromatograms after IgY14-only (a) or tandem IgY14–SuperMix (b) depletion. Overlaid traces showing elution profiles of three reference plasma samples monitored at a 280-nm wavelength over the course of each depletion run. mAU, milli absorbance units.

Figure 3

QC of bRP chromatography and representative bRP chromatograms of iTRAQ- and TMT-labeled plasma peptides. (a–c) Basic pH reversed-phase chromatograms of a standard peptide mixture (a) and plasma (b,c). Mixture consisting of seven peptides is used as a QC of the HPLC system. Sequences of peptides and their elution profiles monitored at 214 nm are shown in a. Typical elution profiles of depleted, digested and iTRAQ- or TMT-labeled plasma monitored at 214 nm are shown in b and c, respectively.

Figure 4

Reproducibility of the plasma processing workflow. Five different aliquots of a pooled plasma sample were depleted using the IgY14–SuperMix tandem depletion strategy and digested. Single 30-μl aliquots of each of the four depletions and two aliquots of the fifth depletion were labeled using TMT6 reagent. After assessing labeling efficiency, the samples were mixed and desalted. The multiplexed sample was fractionated by basic pH reversed-phase chromatography and all 30 fractions were analyzed on a Q Exactive Plus mass spectrometer using in-house packed 75-μm ID picofrit columns packed with 1.9-μm beads to 20-cm length. Data analysis was done using a Spectrum Mill MS Proteomics Workbench. Protein-level total intensity as reported by Spectrum Mill was used for assessing reproducibility. (a) Scatter plots showing correlation of all the replicates. The plot highlighted with the red square is for the two replicates of the same depletion, showing that the correlation is not any different from those for different depletions. (b) Bar and whisker plot of coefficients of variation (CV), showing their median and range.