mTORC1-driven protein translation correlates with clinical benefit of capivasertib within a genetically preselected cohort of PIK3CA-altered tumours.

Capivasertib is a potent selective inhibitor of AKT. It was recently FDA-approved in combination with fulvestrant to treat HR+, HER2-negative breast cancers with certain genetic alteration(s) activating the PI3K pathway. In Phase I trials, heavily pre-treated patients with tumours selected for activating PI3K pathway mutations treated with capivasertib monotherapy demonstrated objective response rates of <30%. We investigated the proteomic profile associated with capivasertib response in genetically pre-selected patients and cancer cell lines. We analyzed samples from 16 PIK3CA-mutated patient tumours collected prior to capivasertib monotherapy in the Phase I trial. PI3K pathway proteins were precisely quantified with immuno-MALDI-MS. Global proteomic profiles were also obtained. Patients were classified according to response to capivasertib monotherapy: "clinical benefit (CB)" (≥12 weeks without progression, n=7) or "no clinical benefit (NCB)" (progression in <12 weeks, n=9). Proteins that differed between the patient groups were subsequently quantified in AKT1- or PIK3CA-altered breast cancer cell lines with varying capivasertib sensitivity. The measured concentrations of AKT1 and AKT2 varied among the PIK3CA-mutated tumours but did not differ between the CB and NCB groups. However, analysis of the global proteome data showed that translational activity was higher in tumours of the NCB vs. CB group. When reproducibly quantified by validated LC-MRM-MS assays, the same proteins of interest similarly distinguished between capivasertib-sensitive vs. -resistant cell lines. The results provide further evidence that increased mTORC1-driven translation functions as a mechanism of resistance to capivasertib monotherapy. Protein concentrations may offer additional insights for patient selection for capivasertib, even among genetically pre-selected patients.

Offline Peptide Fractionation and Parallel Reaction Monitoring MS for the Quantitation of Low-Abundance Plasma Proteins.

Mass spectrometry (MS)-based protein quantitation is an attractive means for research and diagnostics due to its high specificity, precision, sensitivity, versatility, and the ability to develop multiplexed assays for the "absolute" quantitation of virtually any protein target. However, due to the large dynamic range of protein concentrations in blood, high abundance proteins in blood plasma hinder the detectability and quantification of lower-abundance proteins which are often relevant in the context of different diseases. Here we outline a streamlined method involving offline high-pH reversed-phase fractionation of human plasma samples followed by the quantitative analysis of specific fractions using nanoLC-parallel reaction monitoring (PRM) on a Q Exactive Plus mass spectrometer for peptide detection and quantitation with increased sensitivity. Because we use a set of synthetic peptide standards, we can more efficiently determine the precise retention times of the target peptides in the first-dimensional separation and specifically collect eluting fractions of interest for the subsequent targeted MS quantitation, making the analysis faster and easier. An eight-point standard curve was generated by serial dilution of a mixture of previously validated unlabeled ("light") synthetic peptides of interest at known concentrations. The corresponding heavy stable-isotope-labeled standard (SIS) analogues were used as normalizers to account for losses during sample processing and analysis. Using this method, we were able to improve the sensitivity of plasma protein quantitation by up to 50-fold compared to using nanoLC-PRM alone.

Multiple Reaction Monitoring-Mass Spectrometry Enables Robust Quantitation of Plasma Proteins Regardless of Whole Blood Processing Delays That May Occur in the Clinic.

Plasma is an important biofluid for clinical research and diagnostics. In the clinic, unpredictable delays-from minutes to hours-between blood collection and plasma generation are often unavoidable. These delays can potentially lead to protein degradation and modification and might considerably affect intact protein measurement methods such as sandwich enzyme-linked immunosorbent assays that bind proteins on two epitopes to increase specificity, thus requiring largely intact protein structures. Here, we investigated, using multiple reaction monitoring mass spectrometry (MRM-MS), how delays in plasma processing affect peptide-centric "bottom-up" proteomics. We used validated assays for proteotypic peptide surrogates of 270 human proteins to analyze plasma generated after whole blood had been kept at room temperature from 0 to 40 h to mimic delays that occur in the clinic. Moreover, we evaluated the impact of different plasma-thawing conditions on MRM-based plasma protein quantitation. We demonstrate that >90% of protein concentration measurements were unaffected by the thawing procedure and by up to 40-h delayed plasma generation, reflected by relative standard deviations (RSDs) of <30%. Of the 159 MRM assays that yielded quantitative results in 60% of the measured time points, 139 enabled a stable protein quantitation (RSD <20%), 14 showed a slight variation (RSD 20-30%), and 6 appeared unstable/irreproducible (RSD > 30%). These results demonstrate the high robustness and thus the potential for MRM-based plasma-protein quantitation to be used in a clinical setting. In contrast to enzyme-linked immunosorbent assay, peptide-based MRM assays do not require intact three-dimensional protein structures for an accurate and precise quantitation of protein concentrations in the original sample.

A multiplexed, automated immuno-matrix assisted laser desorption/ionization mass spectrometry assay for simultaneous and precise quantitation of PTEN and p110α in cell lines and tumor tissues.

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 the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110α-singleplex iMALDI assays (<15% difference). The multiplexed assay's linear range is from 0.6-20 fmol with accuracies of 90-112% for both target proteins, and the assay is free of matrix-related interferences. The inter-day reproducibility over 5-days was high, with an overall CV of 9%. PTEN and p110α protein concentrations can be quantified down to 1.4 fmol and 0.6 fmol per 10 µg of total tumor protein, respectively, in various tumor tissue samples, including fresh-frozen breast tumors and colorectal cancer liver metastases, and patient-derived xenograft (PDX) tumors.

Performance Assessment of a 125 Human Plasma Peptide Mixture Stored at Room Temperature for Multiple Reaction Monitoring-Mass Spectrometry.

Synthetic peptides are a critical requirement for the development and application of targeted mass spectrometry (MS)-based assays for the quantitation of proteins from biological matrices. Transporting synthetic peptides on dry ice from one laboratory to another is costly and often difficult because of country-specific import and export regulations. Therefore, in this study, we assessed the impact of leaving a lyophilized mixture consisting of 125 peptides at room temperature for up to 20 days, and we assessed the effect on the quantitative performance of multiple reaction monitoring-MS (MRM-MS) assays. The findings suggest that there are no significant differences in the MRM-MS results for the time points assessed in this study (up to 20 days). All the calibration curves and quality control (QC) samples met the acceptance criteria for precision and accuracy (raw data are available via the public MS data repository PanoramaWeb, identifier: /MRM Proteomics/2020_BAK125_RT). The number of endogenous proteins quantifiable across five plasma samples was consistently between 87 and 99 out of 125 for all time points. Moreover, the coefficients of variation (CVs) calculated for the majority of peptide concentrations across all samples and time points were <5%. In addition, a lyophilized peptide mixture was transported from Canada to Iceland without dry ice. The results showed that there was no significant difference in the quantitative performance, with the determined concentrations of most proteins in the samples falling within 30% between the analyses performed on the same three plasma samples in Iceland and those in Canada. Overall, a comparison of the results obtained in Canada and in Iceland indicated that the peptides were stable under the conditions tested and also indicated that shipping lyophilized peptide mixtures without dry ice, but in the presence of sufficient desiccant material, could be a feasible option in cases where transport difficulties may arise or dry-ice sublimation may occur.

Systematic Optimization of the iMALDI Workflow for the Robust and Straightforward Quantification of Signaling Proteins in Cancer Cells.

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.

Immuno-MALDI (iMALDI) mass spectrometry for the analysis of proteins in signaling pathways.

INTRODUCTION: Biomarkers are commonly used to stratify cancer patients and guide targeted therapies, but most biomarkers are of a genomic nature. Discrepancies between the genome and proteome and the high rates of drug resistance indicate that proteomic analyses may provide additional critically important information. Here we present immuno-Matrix-Assisted Laser Desorption/Ionization (iMALDI), the combination of immuno-affinity enrichment of peptides followed by direct MALDI-mass spectrometry analysis. iMALDI is a highly sensitive, targeted protein-quantitation technique with the potential to measure clinically relevant signaling-pathway proteins using minimal sample amounts, thus improving upon existing methodologies. Areas covered: We provide a brief overview of the current state of biomarker analysis technologies for modern cancer treatment. We also show the advantages of iMALDI for translating potential new biomarkers into the clinic, factors to consider for iMALDI assay development, and the utility of iMALDI for the quantitation of cell-signaling proteins. Expert commentary: We see targeted mass spectrometry approaches such as iMALDI as an important part of improving patient responses to targeted therapies by providing highly sensitive, accurate, precise, and specific measurements of signaling-pathway proteins, both in tumor cells and in cells from the tumor microenvironment. iMALDI results can be integrated with other -omics data to aid in tumor-targeting therapies and immuno-oncology.

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

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).

Immuno-Matrix-Assisted Laser Desorption/Ionization Assays for Quantifying AKT1 and AKT2 in Breast and Colorectal Cancer Cell Lines and Tumors.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway is one of the most commonly dysregulated signaling pathways that is linked to cancer development and progression, and its quantitative protein analysis holds the promise to facilitate patient stratification for targeted therapies. Whereas immunohistochemistry (IHC) and immunoassays are routinely used for clinical analysis of signaling pathways, mass spectrometry-based approaches such as liquid chromatography/electrospray ionization multiple reaction monitoring mass spectrometry (LC/ESI-MRM-MS) are more commonly used in clinical research. Both technologies have certain disadvantages, namely, the nonspecificity of IHC and immunoassays, and potentially long analysis times per sample of LC/ESI-MRM-MS. To create a robust, fast, and sensitive protein quantification tool, we developed immuno-matrix-assisted laser desorption/ionization (iMALDI) assays with automated liquid handling. The assays are able to quantify AKT1 and AKT2 from breast cancer and colon cancer cell lines and flash-frozen tumor lysates with a linear range of 0.05-2.0 fmol/µg of total lysate protein and with coefficients of variation < 15%. Compared to other mass spectrometric methods, the developed assays require less sample per analysis-only 25 µg of total protein-and are therefore suitable for analysis of needle biopsies. Furthermore, the presented iMALDI technique is the first MS-based method for absolute quantitation of AKT peptides from cancer tissues. This study demonstrates the suitability of iMALDI for low limit-of-detection and reproducible quantitation of signaling pathway members using a benchtop MALDI mass spectrometer within approximately 6-7 h.