Quantitative proteome analysis of human plasma following in vivo lipopolysaccharide administration using 16O/18O labeling and the accurate mass and time tag approach

Abstract

Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes postdigestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional LC-FTICR mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC elution time AMT tag data base was initially generated using MS/MS following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag data base contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion of high abundance proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses, and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration.

Figures

Figure 1

Strategy for quantitative proteome analysis using 16O/18O labeling and the AMT tag approach. The strategy is a two-stage process. In the initial stage, a peptide AMT tag database for plasma is generated based on extensive analyses of plasma-derived peptide samples using multidimensional LC coupled to tandem mass spectrometry. In the second stage, samples to be compared are separately labeled with 16O and 18O, and then the labeled samples are combined for further SCX fractionation. The fractionated, labeled samples are analyzed by high throughput LC-FTICR and peptide pairs are identified by matching to the AMT tag database and quantified using 18O/16O abundance ratios.

Figure 2

Functional distribution of 938 non-redundant plasma proteins or protein groups (the complete list of identified proteins is available in Supplemental Table 1).

Figure 3

LC-FTICR analysis of 1:1 labeled control plasma samples. (A) A partial 2-D display of the detected 18O/16O labeled peptide pairs. The elution time is shown as a normalized scale between 0 and 1. Observed peaks (represented by spots) correspond to various eluting peptides. The heavy and light isotope-labeled pairs are easily visualized with a 4 Da mass difference. (B) The ratio distribution of 891 detected peptide pairs.

Figure 4

(A) The mass error (left) and NET error (right) distributions of the 3534 identified peptide pairs. For those peptide pairs that were observed across multiple fractions, average mass error and NET error values were used in these plots. (B) Normalized fold changes for the 429 quantified proteins following LPS administration. Abundance ratio for each protein shown was normalized to zero (R – 1). For ratios smaller than 1, normalized inverted ratios were calculated as [1 – (1/R)]. Error bar for each protein indicates the standard deviation for the abundance ratios from multiple peptides. Proteins without error bars were identified with single peptides.

Figure 5

Mass spectra for selected peptide pairs. (A) Two different peptide pairs from C-reactive protein. (B) Three different peptide pairs from Von Willebrand factor.