MSACL 2016 EU Abstract

Multiplex Plasma Protein Analysis: Quantitation of Dozens of Biomarkers from a Droplet of Blood Using Immunoaffinity Mass Spectrometry

Thomas O Joos (Presenter)
NMI Natural and Medical Sciences Institute

Bio: Dr. Thomas Joos joined 1998 the NMI at the University of Tuebingen heading the department of biochemistry. Since 2013 he is the deputy managing director of NMI. His research is focused on miniaturized multiplexed immunoassays for biomarker research and diagnostic applications. Dr. Joos studied Biochemistry at the University of Tubingen. He performed his Ph.D. degree in 1985 on integrin‐alpha5 during early embroygenesis of Xenopus laevis in the laboratory of Prof. Peter Hausen at the Max‐Planck‐Institute of Developmental Biology. Dr. Joos is a member of the editorial board of Drug Discovery Today, Proteomics, Molecular Biotechnology and Expert Review of Proteomics. He is a member of the scientific advisory board of the "Plasma Proteome Institute" Washington, DC, USA,and of Myriad‐RBM, Austin, TX, USA.

Authorship: Lang, B. (1), van den Berg, HJB. (1), Wiese, C. (2), Goepel W. (3), Haertel C. (3), Joos TO. (1), Poetz O. (1)
(1) NMI Natural and Medical Sciences Institute at the University of Tuebingen, (2) Wellspring Clinical Lab, Inc., (3) University Hospital Schleswig Holstein

Short Abstract

Mass spectrometry-based immunoassays enable the measurement of dozens of target proteins from a minute amount of sample. Biological samples like plasma are enzymatically fragmented and peptides derived from proteins of interest are immunoprecipitated by peptide-specific antibodies immobilized on a solid-surface. Indirect quantification could be achieved by using isotopically labelled reference peptides. Here, we present our results for the accurate quantification of more than 12 target proteins in plasma requiring only 5 µl sample material. This low volume meets the assay requirements to monitor plasma proteins in preterm infants to measure plasma parameter relevant to organ development. We quantified those proteins in more than 100 plasma samples from newborns and preterm infants.

Long Abstract

Mass spectrometry-based immunoassays enable the measurement of dozens of target proteins from a minute amount of sample. Usually biological samples like plasma are enzymatically fragmented and peptides derived from proteins of interest are chromatographically separated and analyzed by a mass spectrometer. Indirect protein quantification is achieved by simultaneously measuring a defined, spiked amount of corresponding isotopically-labeled (C13/N15) reference peptide. The sample consumption is very low and a high number of proteins can be analyzed in parallel. Furthermore, mass spectrometric measurements are highly precise. However, the sensitivity of SRM-based protein assays is limited: this is caused e.g. by ion suppression effects that arise from the sample amount and complexity (1) .Peptide-specific antibody sets are used in a parallelized immunoprecipitation step to reduce sample complexity and thereby concurrent suppression effects. As a consequence sensitivity and analytical speed is greatly increased. Although this method does not match the speed and sample throughput of other antibody-based methods, such as sandwich immunoassays, its specificity is excellent (2-4).

This highly specific protein detection is based on two independent lines of evidence: immunoaffinity capture and mass spectrometry based readout. However, broad application of this immunoaffinity MS strategy in proteomic experiments has been restrained by the lack of suitable antibodies. The shortage of specific antibodies is still a limiting factor although rapid progress is being made in large-scale projects targeting the generation of antibodies on a proteomic-wide scale.

Recently we demonstrated that antibodies with a short C-terminal peptide epitope (triple X proteomics antibody) can be applied to enrich peptides that have a common C-terminal motif (5).

In the present study we combined this type of antibody capture with SRM to create a quantitative assay set-up for profiling a variety of plasma parameters, which may reflect liver maturation status of preterm infants. Methods for monitoring multiple plasma protein biomarkers in preterm infants or newborns have not yet been established. Due to the low blood amount (ca. 125 mL) of preterm infants multiplex methods are essential for such biomarker analyses. Routine analysis of plasma proteins would allow monitoring e.g. organ development or inflammation and could prevent severe health damage, consequently improving preterm infants’ overall health.

We developed and applied such MS-based immunoassay for the identification and accurate quantification of 12 plasma proteins generally synthesized in liver requiring only 5 µL plasma. This low volume meets the assay requirements to monitor plasma proteins in preterm infants. We quantified those proteins in more than 100 plasma samples from newborns and preterm infants. This is the first quantitative analysis of protein biomarkers in preterm infants that are relevant to organ development.


References & Acknowledgements:

1. Abbatiello, S. E., Mani, D. R., Keshishian, H. & Carr, S. A. Automated detection of inaccurate and imprecise transitions in peptide quantification by multiple reaction monitoring mass spectrometry. Clin. Chem. 56, 291–305 (2010).

2. Anderson, N. L. et al. Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). J. Proteome Res. 3, 235–244 (2004).

3. Weiß et al. Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry SCIENTIFIC REPORTS 5 : 8759 (2015)

4. Weiß et al. Catch and measure-mass spectrometry-based immunoassays in biomarker research. Biochim Biophys Acta, 1844 (5):927-32. 2014

5. Hoeppe, S. et al. Targeting peptide termini, a novel immunoaffinity approach to reduce complexity in mass spectrometric protein identification. Mol. Cell. Proteomics 10, M110 002857 (2011).


Financial Disclosure

DescriptionY/NSource
GrantsyesMyriad RBM, HOT Screen GmbH
SalaryyesMyriad RBM
Board MemberyesMyriad RBM
Stockno
Expensesno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

no