Mass
spectrometry (MS), particularly targeted proteomics, is increasingly
being used for quantifying specific proteins and peptides in clinical
specimens. The coupling of immuno-enrichment of proteotypic peptides
with MS [e.g., immuno-multiple reaction monitoring (MRM) and immuno-matrix-assisted
laser desorption ionization (MALDI)] enables the development of highly
sensitive and specific assays for low-abundance signaling proteins.
By incorporating stable isotope-labeled standards, these workflows
allow the determination of endogenous protein concentrations. This
is typically achieved through external calibration, often using surrogate
matrices, which has inherent limitations for the analysis of clinical
specimens as there are often substantial variations in the sample
matrix, and sample amounts are typically limited. We have previously
introduced the use of two peptide isotopologues for generating external
calibration curves in plasma. Here, we present a two-point internal
calibration (2-PIC) strategy using two isotopologues for immuno-MS
assays and demonstrate its flexibility and robustness. Quantification
of the tumor suppressor PTEN in Colo-205 cells by immuno-MRM and immuno-MALDI
using 2-PIC and external calibration yielded very similar results
(relative standard deviation between 2-PIC and external calibration:
4.9% for immuno-MRM; 1.1% for immuno-MALDI), without the need for
a surrogate matrix or additional patient material for calibration,
while concurrently reducing the instrument time and cost. Although
our PTEN immuno-MRM and immuno-MALDI assays can be considered to be
orthogonal as they utilized entirely different sample preparation
and MS analysis workflows, targeted different PTEN peptides, and were
performed in different laboratories, the endogenous Colo-205 PTEN
levels determined with 2-PIC showed a good correlation (r
2 = 0.9966) and good agreement (0.48 ± 0.01 and 0.29
± 0.02 fmol/μg of total protein) between immuno-MRM and
immuno-MALDI.
Purpose
Immuno‐MALDI (iMALDI) combines immuno‐enrichment of biomarkers with MALDI‐MS for fast, precise, and specific quantitation, making it a valuable tool for developing clinical assays. iMALDI assays are optimized for the PI3‐kinase signaling pathway members phosphatase and tensin homolog (PTEN) and PI3‐kinase catalytic subunit alpha (p110α), with regard to sensitivity, robustness, and throughput. A standardized template for developing future iMALDI assays, including automation protocols to streamline assay development and translation, is provided.
Experimental Design
Conditions for tryptic digestion and immuno‐enrichment (beads, bead:antibody ratios, incubation times, direct vs. indirect immuno‐enrichment) are rigorously tested. Different strategies for calibration and data readout are compared.
Results
Digestion using 1:2 protein:trypsin (wt:wt) for 1 h yielded high and consistent peptide recoveries. Direct immuno‐enrichment (antibody‐bead coupling prior to antigen‐enrichment) yielded 30% higher peptide recovery with a 1 h shorter incubation time than indirect enrichment. Immuno‐enrichment incubation overnight yielded 1.5‐fold higher sensitivities than 1 h incubation. Quantitation of the endogenous target proteins is not affected by the complexity of the calibration matrix, further simplifying the workflow.
Conclusions and Clinical Relevance
This optimized and automated workflow will facilitate the clinical translation of high‐throughput sensitive iMALDI assays for quantifying cell‐signaling proteins in individual tumor samples, thereby improving patient stratification for targeted treatment.
The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of...
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