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

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).

Broad‐Spectrum Antiviral Peptides and Polymers

“…Although peptides have narrow molecular weight distribution and can be readily quantified in the plasma using MALDI or LC-MS to study pharmacokinetics, they have shortcomings of high cost and short blood circulation time due to enzymatic degradation. [211,212] These issues may be overcome by rigidifying the structure of peptides, as mentioned in Section 2.2. [74,75,78] Antiviral material research would progress rapidly with standardization efforts among different laboratories as this would allow the use of machine learning to accelerate the development of broad-spectrum antiviral macromolecules against emerging or re-emerging viruses such as the SARS-CoV-2 virus.…”

Detecting post-translational modification signatures as potential biomarkers in clinical mass spectrometry

“…If 90% of the non-modified background can be removed, a larger proportion of the PTM-proteome of interest can be loaded on-column to improve sensitivity. If this is still not sufficient, combining anti-peptide antibody immunoprecipitation (IP) with MS, such as 'Stable Isotope Standards and Capture by Anti-Peptide Antibodies' (SISCAPA) [105][106][107] and immuno MALDI (iMALDI) [108][109][110][111], or TXP antibodies [112] that recognize peptides with a common epitope of 3-4 amino acids at the N-or C-terminus, prior to (LC-)MS analysis can considerably improve the accessibility of target peptides. The unique capabilities of MS can even compensate for low antibody Figure 5.…”

“…Translation to clinical settings requires robust methods that are precise, cheap and provide high throughput, among those immunoassays (given the previously mentioned limitations), targeted MS with robust LC conditions, as well as SISCAPA [105][106][107] and iMALDI [108][109][110][111].…”