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

Summary

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

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).

Introduction

Mass spectrometry has become an indispensable tool in quantitative proteomics. Mass spectrometry can determine the masses of target proteins or peptides, therefore the obtained analyte signals can be highly specific compared to immunoassays. Two ionization methods, electrospray and MALDI, are most commonly used for detecting proteins and peptides^1,2,3,4. A major challenge in MS-based protein quantification lies in the detection of low-abundance proteins in complex samples at ng/mL or pg/mL concentrations in the presence of high-abundance pro....

Protocol

The amounts of the reagents described below are based on the measurement of 20 patient plasma samples. The protocol presented below follows the guidelines of the University of Victoria's human research ethics committee.

1. Generation of Ang I in Human Plasma

1. Thaw plasma samples (≥200 µL) in a room temperature water bath for 5 min, and then put the samples on ice until completely thawed.
2. Transfer 200 µL of each plasma sample manually to separate wells of a 1.1 mL deep-well plate (sample plate), and centrifuge the plate in a centrifuge for 10 min at 2 °C and 1278 x g.
3. Use an automated l....

Results

An automated iMALDI procedure for measuring Ang I is shown in Figure 1. Target peptides (either endogenous peptides or peptides from digested proteins) are enriched on anti-peptide magnetic beads, and then the beads are transferred to a target plate for MALDI measurement. The whole procedure is simplified compared to other immuno-MS technologies that require additional peptide elution steps. Automation of the iMALDI assay allows for high-throughput analysis o.......

Discussion

Compared to conventional MS-based protein quantification, iMALDI uses antibodies to enrich the analytes and purify them from complex samples, therefore making it possible to quantify proteins or peptides at low concentrations. A critical step in the iMALDI protocol is the immuno-enrichment of the target peptides. For this purpose, antibodies with high specificity and affinity should be selected. In SISCAPA, it has been reported that antibody affinities at 10^-9 M or better would be needed to achieve high sensit.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Healthy control human plasma	Bioreclamation	HMPLEDTA2
Ammonium bicarbonate	Sigma Aldrich	09830
Ammonium citrate dibasic	Sigma Aldrich	09833
CHAPS (>=98%)	Sigma Aldrich	C9426
Albumin from chicken egg white (>98%)	Sigma Aldrich	A5503
Ethylenediaminetetraacetic acid	Sigma Aldrich	EDS
Alpha-cyano-4-hydroxycinnamic acid	Sigma Aldrich	70990
Phosphate buffered saline	Sigma Aldrich	P4417
Phenylmethanesulfonyl fluoride	Sigma Aldrich	78830
Trifluoroacetic acid	Thermo Fisher Scientific	85172	LC-MS grade
acetonitrile	Fluka	34967	LC-MS grade
water	Fluka	39253	LC-MS grade
acetic acid	Fluka	320099	LC-MS grade
Tris(hydroxymethyl)aminomethane	Roche Diagnostics	3118169001
Dynabeads Protein G magnetic beads	Thermo Fisher Scientific	10003D	2.8 μm, 30 mg/mL
anti-Ang I goat polyclonal antibody	Santa Cruz Biotechnology	sc-7419
Nat and SIS Ang I	synthesized at the University of Victoria-Genome BC Proteomics Centre
Automated liquid handling system	Agilent	16050-102	Agilent Bravo robotic workstation
Magnet	Thermo Fisher Scientific	12321D	Invitrogen DynaMag-2 magnet
Tube rotator	Theromo Scientific	400110Q	Labquake Tube Rotator
Magnet	Thermo Fisher Scientific	12027	DynaMag-96 side skirted magnet
Magnet	VP Scientific	771RM-1	used to pull the beads to the bottom of the well
MALDI-TOF	Bruker		Bruker Microflex LRF instrument