Sequential multiplexed analyte quantification using peptide immunoaffinity enrichment coupled to mass spectrometry.
Peptide immunoaffinity enrichment coupled to selected reaction monitoring (SRM) mass spectrometry (immuno-SRM) has emerged as a technology with great potential for quantitative proteomic assays. One advantage over traditional immunoassays is the tremendous potential for concurrent quantification of multiple analytes from a given sample (i.e. multiplex analysis). We sought to explore the capacity of the immuno-SRM technique for analyzing large numbers of analytes by evaluating the multiplex capabilities and demonstrating the sequential analysis of groups of peptides from a single sample. To evaluate multiplex analysis, immuno-SRM assays were arranged in groups of 10, 20, 30, 40, and 50 peptides using a common set of reagents. The multiplex immuno-SRM assays were used to measure synthetic peptides added to plasma covering several orders of magnitude concentration. Measurements made in large multiplex groups were highly correlated (r(2) ≥ 0.98) and featured good agreement (bias ≤ 1%) compared with single-plex assays or a 10-plex configuration. The ability to sequentially enrich sets of analyte peptides was demonstrated by enriching groups of 10 peptides from a plasma sample in a sequential fashion. The data show good agreement (bias ≤ 1.5%) and similar reproducibility regardless of enrichment order. These significant advancements demonstrate the utility of immuno-SRM for analyzing large numbers of analytes, such as in large biomarker verification experiments or in pathway-based targeted analysis.
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Jeffrey R
JR
Whiteaker
Lei
L
Zhao
Chenwei
C
Lin
Ping
P
Yan
Pei
P
Wang
Amanda G
AG
Paulovich
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D006801:Humans; D007163:Immunosorbent Techniques; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D010449:Peptide Mapping; D010949:Plasma / Q000737:chemistry; D040901:Proteomics; D015203:Reproducibility of Results
D010446:Peptide Fragments