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J Vis Exp. 2017 Aug 18;(126). doi: 10.3791/55933.

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI).

Author information

1
University of Victoria-Genome BC Proteomics Centre.
2
Jewish General Hospital, McGill University.
3
University of Victoria-Genome BC Proteomics Centre; Dept of Biochemistry and Microbiology, University of Victoria; Proteomics Centre, Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University; Gerald Bronfman Department of Oncology, Jewish General Hospital; christoph@proteincentre.com.

Abstract

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

PMID:
28872133
PMCID:
PMC5614345
[Available on 2019-08-18]
DOI:
10.3791/55933
[Indexed for MEDLINE]

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