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


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.

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Figure 1: Overview of multiplexed workflow for discovery proteomics in plasma.
Figure 2: Chromatogram of plasma after immunoaffinity depletion.
Figure 3: QC of bRP chromatography and representative bRP chromatograms of iTRAQ- and TMT-labeled plasma peptides.
Figure 4: Reproducibility of the plasma processing workflow.


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We thank N. Udeshi for reading the manuscript and providing valuable feedback. This work was supported in part by grants from the National Institutes of Health: HHSN268201000033C and R01HL096738 from the National Heart, Lung, and Blood Institute (NHLBI; to S.A.C.) and grants U24CA160034 from the National Cancer Institute (NCI) Clinical Proteomics Tumor Analysis Consortium initiative and U01CA152990 from the NCI Early Detection Research Network program (to M.A.G.).

Author information




H.K., M.W.B., H.S., K.R.C., M.A.G. and S.A.C. developed the protocol. L.W., M.W.B. and H.S. optimized and ran QC checks on many aspects of the protocol. H.K., M.W.B., H.S., K.R.C., M.A.G. and S.A.C. wrote the manuscript.

Corresponding authors

Correspondence to Hasmik Keshishian or Steven A Carr.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Plumbing schematic for IgY14-Supermix tandem depletion columns on an Agilent 1200 LC system.

Diagram illustrates plumbing the second valve for tandem setup of 2 depletion columns. A 2mL needle seat extension has been incorporated to accommodate injections of more than 900μL on the Agilent 1200 system.

Supplementary Figure 2 Chromatograms of plasma after tandem IgY14-Supermix depletion demonstrating column performance over the vendor recommended lifetime.

Injection number 7(blue), 49 (red) and 100 (green) are overlaid showing a reduction in peak area of the IgY14 bound peak with a concomitant increase and change in shape of the IgY14-Supermix depleted peak. In addition, an early peak (1) is also observed by injection 49 increasing over the remainder of the column lifetime. We estimate that area ratio of IgY14-Supemix depleted peak to IgY14 bound peak should be around 0.06-0.07 for effective depletion. As column ages this ratio increases and ratio of more than 0.1 indicates problematic depletion.

Supplementary Figure 3 Summary statistics in four PMI patient samples (Adapted from (38)).

Table gives details about peptide and protein identification and quantitation in each of the patient sample. Venn diagram shows the overlap in between the four patient samples. aProteins identified in at least two patients with two or more peptides. bSubset of identified proteins with two or more distinct peptides observed in at least one patient. cProtein subgroups (groups); that is, 5304 distinct protein subgroups were identified within 4555 protein groups. Proteins that share a detected distinct peptide (length > 8) are combined into a group. A protein group is parsimoniously expanded to one or more subgroups to distinguish proteins that also have one or more distinct peptides that are not shared with the rest of the group, typically isoforms and family members.

Supplementary Figure 4 Timelines and throughput for (A) plasma sample processing and (B) LC-MS/MS data collection.

Times for each processing step are illustrated for deep plasma profiling employing iTRAQ4-plex and TMT10-plex modes with off-line basic reversed phase peptide fractionation, as well as for single shot (no off-line fractionation) analysis of peptides labeled with TMT10. Times shown are those required for a single person manually processing samples. All sample processing steps can be further parallelized, increasing the throughput, making LC-MS/MS analysis on a single instrument the rate-limiting step of the deep profiling workflow (B). In contrast, in the single shot analysis workflow 4 different plexes (16 – 40 samples, depending on reagent used for labeling) can be analyzed on a single LC-MS/MS instrument in a single day making sample processing the limiting factor for this workflow.

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Keshishian, H., Burgess, M., Specht, H. et al. Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry. Nat Protoc 12, 1683–1701 (2017). https://doi.org/10.1038/nprot.2017.054

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