Acoustic ejection mass spectrometry empowers ultra-fast protein biomarker quantification.

The global scientific response to COVID 19 highlighted the urgent need for increased throughput and capacity in bioanalytical laboratories, especially for the precise quantification of proteins that pertain to health and disease. Acoustic ejection mass spectrometry (AEMS) represents a much-needed paradigm shift for ultra-fast biomarker screening. Here, a quantitative AEMS assays is presented, employing peptide immunocapture to enrich (i) 10 acute phase response (APR) protein markers from plasma, and (ii) SARS-CoV-2 NCAP peptides from nasopharyngeal swabs. The APR proteins were quantified in 267 plasma samples, in triplicate in 4.8 h, with %CV from 4.2% to 10.5%. SARS-CoV-2 peptides were quantified in triplicate from 145 viral swabs in 10 min. This assay represents a 15-fold speed improvement over LC-MS, with instrument stability demonstrated across 10,000 peptide measurements. The combination of speed from AEMS and selectivity from peptide immunocapture enables ultra-high throughput, reproducible quantitative biomarker screening in very large cohorts.

A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

Virome-wide detection of natural infection events and the associated antibody dynamics using longitudinal highly-multiplexed serology.

Current methods for detecting infections either require a sample collected from an actively infected site, are limited in the number of agents they can query, and/or yield no information on the immune response. Here we present an approach that uses temporally coordinated changes in highly-multiplexed antibody measurements from longitudinal blood samples to monitor infection events at sub-species resolution across the human virome. In a longitudinally-sampled cohort of South African adolescents representing >100 person-years, we identify >650 events across 48 virus species and observe strong epidemic effects, including high-incidence waves of Aichivirus A and the D68 subtype of Enterovirus D earlier than their widespread circulation was appreciated. In separate cohorts of adults who were sampled at higher frequency using self-collected dried blood spots, we show that such events temporally correlate with symptoms and transient inflammatory biomarker elevations, and observe the responding antibodies to persist for periods ranging from ≤1 week to >5 years. Our approach generates a rich view of viral/host dynamics, supporting novel studies in immunology and epidemiology.

Cov2MS: An Automated and Quantitative Matrix-Independent Assay for Mass Spectrometric Measurement of SARS-CoV-2 Nucleocapsid Protein.

The pandemic readiness toolbox needs to be extended, targeting different biomolecules, using orthogonal experimental set-ups. Here, we build on our Cov-MS effort using LC-MS, adding SISCAPA technology to enrich proteotypic peptides of the SARS-CoV-2 nucleocapsid (N) protein from trypsin-digested patient samples. The Cov2MS assay is compatible with most matrices including nasopharyngeal swabs, saliva, and plasma and has increased sensitivity into the attomole range, a 1000-fold improvement compared to direct detection in a matrix. A strong positive correlation was observed with qPCR detection beyond a quantification cycle of 30-31, the level where no live virus can be cultured. The automatable sample preparation and reduced LC dependency allow analysis of up to 500 samples per day per instrument. Importantly, peptide enrichment allows detection of the N protein in pooled samples without sensitivity loss. Easily multiplexed, we detect variants and propose targets for Influenza A and B detection. Thus, the Cov2MS assay can be adapted to test for many different pathogens in pooled samples, providing longitudinal epidemiological monitoring of large numbers of pathogens within a population as an early warning system.

Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Development of an automated, interference-free, 2D-LC-MS/MS assay for quantification of a therapeutic mAb in human sera.

AIM: Hybrid LC-MS/MS assays are increasingly used to quantitate proteins in biological matrices. These assays involve analyte enrichment at the protein level. Although suitability has been demonstrated, they are limited by the lack of appropriate affinity reagents and may suffer from interferences caused by binding proteins or antibodies. RESULTS: An online stable isotope standards and capture by anti-peptide antibodies assay was developed, which involves tryptic digestion of a therapeutic monoclonal antibody in human serum to destroy interfering proteins followed by enrichment using high affinity peptide antibodies. The assay was validated and compared with a standard ligand-binding assay currently used for quantification. CONCLUSION: The data show that the stable isotope standards and capture by anti-peptide antibodies-2D-LC-MS/MS assay can be used as an alternative method for measurement of monoclonal antibodies in clinical samples.

Measurement of Lipoprotein-Associated Phospholipase A2 by Use of 3 Different Methods: Exploration of Discordance between ELISA and Activity Assays.

BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme associated with inflammation, is used as a biomarker for cardiovascular disease risk. Both the concentration and activity of Lp-PLA2 have been shown to be clinically relevant. However, there is a discordance between the serum concentration of Lp-PLA2 measured by the standard ELISA-based immunoassays and the activity of this enzyme, leading to substantial discordance in risk categorization depending on assay format. METHODS: We developed 2 LC-MS/MS-based assays to quantify serum Lp-PLA2 activity (multiple reaction monitoring detection of product) and concentration [stable isotope standards and capture by antipeptide antibody (SISCAPA) immunoaffinity], and we investigated their correlation to commercially offered colorimetric activity and immunometric concentrations assays. Associations between Lp-PLA2 and lipoproteins and the effect of selected detergents in liberating Lp-PLA2 were evaluated by use of immunoprecipitation and Western blot analyses. RESULTS: Serum Lp-PLA2 concentrations measured by quantitative SISCAPA-mass spectrometry were substantially higher than concentrations typically measured by immunoassay and showed an improved agreement with Lp-PLA2 activity. With detergents, liberation of Lp-PLA2 from lipoprotein complexes dramatically increased the amount of protein detected by immunoassay and improved the agreement with activity measurements. CONCLUSIONS: Quantitative analysis of Lp-PLA2 concentration and activity by LC-MS/MS assays provided key insight into resolving the well-documented discordance between Lp-PLA2 concentration (determined by immunoassay) and activity. Quantitative detection of Lp-PLA2 by immunoassay appears to be strongly inhibited by interaction of Lp-PLA2 with lipoprotein. Together, the results illustrate the advantages of quantitative LC-MS/MS for measurement of Lp-PLA2 concentration (by SISCAPA) and activity (by direct product detection).

Multiplexed longitudinal measurement of protein biomarkers in DBS using an automated SISCAPA workflow.

BACKGROUND: The use of DBS for quantitative protein biomarker measurement has been hindered by issues associated with blood hematocrit variations and lack of detection sensitivity, particularly when multiple biomarkers are measured. MATERIALS & METHODS: An automated, multiplexed SISCAPA analysis was used to normalize blood volume variations in DBS and quantify proteins of varying abundance in longitudinal specimens. CONCLUSION: The results showed that after normalizing the spot-to-spot hematocrit variations, peptide surrogates of protein biomarkers could be accurately quantitated in DBS. This allowed the establishment of baselines for a variety of biomarkers in multiple individuals and enabled detection of changes over time, thus offering an effective solution for longitudinal personal monitoring of biomarkers relevant in health and disease.

Clinical translation of MS-based, quantitative plasma proteomics: status, challenges, requirements, and potential.

INTRODUCTION: Aided by the advent of advanced mass spectrometry (MS)-based technologies and methodologies, quantitative proteomics has emerged as a viable technique to capture meaningful data for candidate biomarker evaluation. To aid clinical translation, these methods generally utilize a bottom-up strategy with isotopically labeled standards and a targeted form of MS measurement. AREAS COVERED: This article reviews the status, challenges, requirements, and potential of translating current, MS-based methods to the clinical laboratory. The described methods are discussed and contrasted within a fit-for-purpose approach, while different resources for quality control, quantitative analysis, and data interpretation are additionally provided. Expert commentary: Although great strides have been made over the past five years in developing reliable quantitative assays for plasma protein biomarkers, it is crucial for investigators to have an understanding of the clinical validation process, a major roadblock in translational research. Continued progress in method design and validation of protein assays is necessary to ultimately achieve widespread adoption and regulatory approval.

Quantification of serum apolipoproteins A-I and B-100 in clinical samples using an automated SISCAPA-MALDI-TOF-MS workflow.

A fully automated workflow was developed and validated for simultaneous quantification of the cardiovascular disease risk markers apolipoproteins A-I (apoA-I) and B-100 (apoB-100) in clinical sera. By coupling of stable-isotope standards and capture by anti-peptide antibodies (SISCAPA) for enrichment of proteotypic peptides from serum digests to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS detection, the standardized platform enabled rapid, liquid chromatography-free quantification at a relatively high throughput of 96 samples in 12h. The average imprecision in normo- and triglyceridemic serum pools was 3.8% for apoA-I and 4.2% for apoB-100 (4 replicates over 5 days). If stored properly, the MALDI target containing enriched apoA-1 and apoB-100 peptides could be re-analyzed without any effect on bias or imprecision for at least 7 days after initial analysis. Validation of the workflow revealed excellent linearity for daily calibration with external, serum-based calibrators (R(2) of 0.984 for apoA-I and 0.976 for apoB-100 as average over five days), and absence of matrix effects or interference from triglycerides, protein content, hemolysates, or bilirubins. Quantification of apoA-I in 93 normo- and hypertriglyceridemic clinical sera showed good agreement with immunoturbidimetric analysis (slope = 1.01, R(2) = 0.95, mean bias = 4.0%). Measurement of apoB-100 in the same clinical sera using both methods, however, revealed several outliers in SISCAPA-MALDI-TOF-MS measurements, possibly as a result of the lower MALDI-TOF-MS signal intensity (slope = 1.09, R(2) = 0.91, mean bias = 2.0%). The combination of analytical performance, rapid cycle time and automation potential validate the SISCAPA-MALDI-TOF-MS platform as a valuable approach for standardized and high-throughput quantification of apoA-I and apoB-100 in large sample cohorts.

Serum concentrations of prostate-specific antigen measured using immune extraction, trypsin digestion, and tandem mass spectrometry quantification of LSEPAELTDAVK peptide.

CONTEXT: Prostate-specific antigen (PSA) is a 34-kDa glycoprotein with chymotrypsin-like enzyme activity that circulates both in free forms and complexed to various enzyme inhibitors including antichymotrypsin and α2-macroglobulin. Prostate-specific antigen bound to α2-macroglobulin is not detected by commercial PSA immunoassays. OBJECTIVE: To develop a mass spectrometry assay that detects the same forms of PSA as the immunoassays, which could serve as a reference for harmonizing PSA immunoassays. DESIGN: Prostate-specific antigen was immune extracted from serum, trypsin was digested, and the LSEPAELTDAVK peptide was quantitated on an API 5000 spectrometer. Calibrators were made by adding 10% free and 90% antichymotrypsin-bound PSA to female sera. The assay was standardized to the World Health Organization 96/670 reference standard. Validation of clinical utility and comparisons with 2 immunoassays (Roche cobas and Beckman Access) were performed using frozen sera aliquots from 100 men undergoing prostate biopsy (50 negative, 50 with cancer) and 5 serial samples collected over time from 5 men with advanced prostate cancer. RESULTS: The antibody extraction efficiency was greater than 99%. The assay has an analytic range from 1.2 to 76 ng/mL, with precision ranging from 8.6% at 1.5 ng/mL to 5.4% at 27 ng/mL. The mass spectrometry assay correlated well with 2 immunoassays. All 3 assays showed statistically equivalent separation of prostate cancer from benign disease using receiver operating characteristic curve analysis. CONCLUSIONS: This mass spectrometry assay can reliably measure PSA concentrations in human serum and could serve as a reference standard for harmonizing PSA immunoassays.

Mass spectrometry measurements of prostate-specific antigen (PSA) peptides derived from immune-extracted PSA provide a potential strategy for harmonizing immunoassay differences.

OBJECTIVES: Harmonization of prostate-specific antigen (PSA) immunoassays is important for good patient care. The specificity of the antibodies used to detect circulating PSA could cause differences in the PSA measurements. METHODS: We used mass spectrometry (MS) to quantitate the concentration of five peptides cleaved from trypsin digestion of PSA and compared these measurements with six automated immunoassays. Linear regression and a mixed-effects model were used to analyze the results. RESULTS: PSA measurements from the immunoassays and the five MS peptide assays were highly correlated (R(2) > 0.99), but the recovery of the World Health Organization standard and the regression slopes differed across assays. The same relative patterns of immunoassay differences were seen in comparing their results with each of the five MS peptide measurements from different parts of the circulating PSA molecules. CONCLUSIONS: Mass spectrometry quantitation of peptides derived from trypsin digestion of immune-extracted PSA could be used to harmonize PSA immunoassays.

Quantification of a proteotypic peptide from protein C inhibitor by liquid chromatography-free SISCAPA-MALDI mass spectrometry: application to identification of recurrence of prostate cancer.

BACKGROUND: Biomarker validation remains one of the most challenging constraints to the development of new diagnostic assays. To facilitate biomarker validation, we previously developed a chromatography-free stable isotope standards and capture by antipeptide antibodies (SISCAPA)-MALDI assay allowing rapid, high-throughput quantification of protein analytes in large sample sets. Here we applied this assay to the measurement of a surrogate proteotypic peptide from protein C inhibitor (PCI) in sera from patients with prostate cancer. METHODS: A 2-plex SISCAPA-MALDI assay for quantification of proteotypic peptides from PCI and soluble transferrin receptor (sTfR) was used to measure these peptides in 159 trypsin-digested sera collected from 51 patients with prostate cancer. These patients had been treated with radiation with or without neoadjuvant androgen deprivation. RESULTS: Patients who experienced biochemical recurrence of prostate cancer showed decreased serum concentrations of the PCI peptide analyte within 18 months of treatment. The PCI peptide concentrations remained increased in the sera of patients who did not experience cancer recurrence. Prostate-specific antigen concentrations had no predictive value during the same time period. CONCLUSIONS: The high-throughput, liquid chromatography-free SISCAPA-MALDI assay is capable of rapid quantification of proteotypic PCI and sTfR peptide analytes in complex serum samples. Decreased serum concentrations of the PCI peptide were found to be related to recurrence of prostate cancer in patients treated with radiation with or without hormone therapy. However, a larger cohort of patients will be required for unequivocal validation of the PCI peptide as a biomarker for clinical use.

High-throughput SISCAPA quantitation of peptides from human plasma digests by ultrafast, liquid chromatography-free mass spectrometry.

We investigated the utility of an SPE-MS/MS platform in combination with a modified SISCAPA workflow for chromatography-free MRM analysis of proteotypic peptides in digested human plasma. This combination of SISCAPA and SPE-MS/MS technology allows sensitive, MRM-based quantification of peptides from plasma digests with a sample cycle time of ∼7 s, a 300-fold improvement over typical MRM analyses with analysis times of 30-40 min that use liquid chromatography upstream of MS. The optimized system includes capture and enrichment to near purity of target proteotypic peptides using rigorously selected, high affinity, antipeptide monoclonal antibodies and reduction of background peptides using a novel treatment of magnetic bead immunoadsorbents. Using this method, we have successfully quantitated LPS-binding protein and mesothelin (concentrations of ∼5000 ng/mL and ∼10 ng/mL, respectively) in human plasma. The method eliminates the need for upstream liquid-chromatography and can be multiplexed, thus facilitating quantitative analysis of proteins, including biomarkers, in large sample sets. The method is ideal for high-throughput biomarker validation after affinity enrichment and has the potential for applications in clinical laboratories.

Precision of heavy-light peptide ratios measured by maldi-tof mass spectrometry.

We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100 000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R(2) values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14 000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable currently impractical large-scale verification studies of protein biomarkers.

Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma.

The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the interlaboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay coefficient of variance, limit of detection, limit of quantification, and recovery was evaluated. Limits of detection were at or below 1 ng/ml for the assayed proteins in 30 µl of plasma. Assay reproducibility was acceptable for verification studies, with median intra- and interlaboratory coefficients of variance above the limit of quantification of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope-labeled protein as an internal standard instead of stable isotope-labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-MRM-MS can be made reproducible across independent laboratories and has the potential to be adopted widely for assaying proteins in matrices as complex as plasma.

Quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').(1) An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).(2) In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules (3, 4) and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.(5-7) To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.(8-13) In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.