An innovative standard for LC-MS-based HCP profiling and accurate quantity assessment: Application to batch consistency in viral vaccine samples.

Biotherapeutics, molecules produced from biological systems, require rigorous purification steps to remove impurities including host cell proteins (HCPs). Regulatory guidelines require manufacturers to monitor process-related impurities along the purification workflow. Mass spectrometry (MS) has recently been considered as a complementary method to the well-established ELISA for HCPs quantification, since it has the advantage of unambiguously identifying individual HCP. In this study, we developed an innovative standard dedicated to MS-based HCP profiling analysis in order to monitor the consistency of viral vaccine intermediate purification samples. This standard, termed the HCP-PROFILER standard, is composed of a water-soluble bead (READYBEADS technology) which, after being added into the sample, releases unlabeled peptides in controlled amounts. The standard meets three desired criteria: (1) it is composed of multiple peptides, at different concentration levels, allowing construction of a calibration curve covering the dynamic range of HCPs present in the target sample, ensuring quantification accuracy; (2) it demonstrates high batch-to-batch reproducibility, ensuring quantification robustness and consistency over time; and (3) it is easy to use and avoids user-induced analytical biases. In this study, we present the use of the HCP-PROFILER standard for vaccine batches comparison and downstream process performance studies.

Early and specific targeted mass spectrometry-based identification of bacteria in endotracheal aspirates of patients suspected with ventilator-associated pneumonia.

Rapid and reliable pathogen identification is compulsory to confirm ventilator-associated pneumonia (VAP) in order to initiate appropriate antibiotic treatment. In the present proof of concept, the effectiveness of rapid microorganism identification with a targeted bottom-up proteomics approach was investigated in endotracheal aspirate (ETA) samples of VAP patients. To do so, a prototype selected-reaction monitoring (SRM)-based assay was developed on a triple quadrupole mass spectrometer tracking proteotypic peptide surrogates of bacterial proteomes. Through the concurrent monitoring of 97 species-specific peptides, this preliminary assay was dimensioned to characterize the occurrence of six most frequent bacterial species responsible for over more than 65% of VAP. Assay performance was subsequently evaluated by analyzing early and regular 37 ETA samples collected from 15 patients. Twenty-five samples were above the significant threshold of 105 CFU/mL and five samples showed mixed infections (both pathogens ≥ 105 CFU/mL). The targeted proteomics assay showed 100% specificity for Acinetobacter baumannii, Escherichia coli, Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. No false bacterial identification was reported and no interference was detected arising from the commensal flora. The overall species identification sensitivity was 19/25 (76%) and was higher at the patient level (84.6%). This successful proof of concept provides a rational to broaden the panel of bacteria for further clinical evaluation.

Streamlined Development of Targeted Mass Spectrometry-Based Method Combining Scout-MRM and a Web-Based Tool Indexed with Scout Peptides.

MS-based targeted proteomics is a relevant technology for sensitive and robust relative or absolute quantification of proteins biomarker candidates in complex human biofluids or tissue extracts. Performing a multiplex assay imposes time scheduling of peptide monitoring only around their expected retention time that needs to be defined with synthetic peptide. Time-scheduled monitoring is clearly a constraint that precludes from straightforward assay transfer between biological matrices or distinct experimental setup. Any unexpected retention time (RT) shift challenges assay robustness and its implementation for large-scale analysis. Recently, Scout-multiple reaction monitoring that fully releases multiplexed targeted acquisition from RT scheduling by successively monitoring complex transition groups triggered with sentinel molecules called Scout has been introduced. It is herein documented how Peptide Selector database and tool streamlines the building of a multiplexed method thanks to RT indexation relative to Scout peptides. This case study deals with surrogate peptides of biomarker candidates related to drug-induced liver and vascular injury, running such on-line built method (eight Scouts triggering the monitoring of a total of 692 transitions) enables 100% recovery of a panel of 93 spiked-in heavy labeled standards, despite significant RT shifts between serum, plasma, or urine. This result illustrates the simplicity of automatically building and deploying robust proteomics targeted assay.

Variance component analysis to assess protein quantification in biomarker validation: application to selected reaction monitoring-mass spectrometry.

BACKGROUND: In the field of biomarker validation with mass spectrometry, controlling the technical variability is a critical issue. In selected reaction monitoring (SRM) measurements, this issue provides the opportunity of using variance component analysis to distinguish various sources of variability. However, in case of unbalanced data (unequal number of observations in all factor combinations), the classical methods cannot correctly estimate the various sources of variability, particularly in presence of interaction. The present paper proposes an extension of the variance component analysis to estimate the various components of the variance, including an interaction component in case of unbalanced data. RESULTS: We applied an experimental design that uses a serial dilution to generate known relative protein concentrations and estimated these concentrations by two processing algorithms, a classical and a more recent one. The extended method allowed estimating the variances explained by the dilution and the technical process by each algorithm in an experiment with 9 proteins: L-FABP, 14.3.3 sigma, Calgi, Def.A6, Villin, Calmo, I-FABP, Peroxi-5, and S100A14. Whereas, the recent algorithm gave a higher dilution variance and a lower technical variance than the classical one in two proteins with three peptides (L-FABP and Villin), there were no significant difference between the two algorithms on all proteins. CONCLUSIONS: The extension of the variance component analysis was able to estimate correctly the variance components of protein concentration measurement in case of unbalanced design.

Impact of Serum and Plasma Matrices on the Titration of Human Inflammatory Biomarkers Using Analytically Validated SRM Assays.

Protein biomarker discovery has inherent challenges linked to the validation of the analytical method used or to the impact of biological matrices. Matrix influences must be mastered to guarantee the reliability of the identified biomarkers to monitor human diseases. In this study, multiplexed mass spectrometry assays in selected reaction monitoring (SRM) mode have been developed to measure 107 inflammatory putative proteins in matched serum and plasma from 36 ICU trauma patients. The assays' validation directly in clinical samples was shown to be valuable to manage intersample variability. Using the validation process developed here, assays were validated for 58 biomarkers in serum, 57 in plasma, and 55 in both matrices. Correlation analyses demonstrated that the quantitation using SRM of most of the validated biomarkers (45/55) was impacted by the biological matrix and that the matrix impact was biomarker-dependent. Among the 45 impacted biomarkers, 23 were nevertheless correlated between serum and plasma, whereas the quantitation was shown to be equivalent in both for the 10 last proteins. Matrix selection using SRM is therefore suggested to be suitable prior to clinical evaluation of biomarkers in a large cohort of patients.

Clinical quantitation of prostate-specific antigen biomarker in the low nanogram/milliliter range by conventional bore liquid chromatography-tandem mass spectrometry (multiple reaction monitoring) coupling and correlation with ELISA tests.

Proteomics discovery leads to a list of potential protein biomarkers that have to be subsequently verified and validated with a statistically viable number of patients. Although the most sensitive, the development of an ELISA test is time-consuming when antibodies are not available and need to be conceived. Mass spectrometry analysis driven in quantitative multiple reaction monitoring mode is now appearing as a promising alternative to quantify proteins in biological fluids. However, all the studies published to date describe limits of quantitation in the low microg/ml range when no immunoenrichment of the target protein is applied, whereas the concentration of known clinical biomarkers is usually in the ng/ml range. Using prostate-specific antigen as a model biomarker, we now provide proof of principle that mass spectrometry enables protein quantitation in a concentration range of clinical interest without immunoenrichment. We have developed and optimized a robust sample processing method combining albumin depletion, trypsin digestion, and solid phase extraction of the proteotypic peptides starting from only 100 microl of serum. For analysis, mass spectrometry was coupled to a conventional liquid chromatography system using a 2-mm-internal diameter reverse phase column. This mass spectrometry-based strategy was applied to the quantitation of prostate-specific antigen in sera of patients with either benign prostate hyperplasia or prostate cancer. The quantitation was performed against an external calibration curve by interpolation, and results showed good correlation with existing ELISA tests applied to the same samples. This strategy might now be implemented in any clinical laboratory or certified company for further evaluation of any putative biomarker in the low ng/ml range of serum or plasma.

Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry.

Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60-80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.

The current status of clinical proteomics and the use of MRM and MRM(3) for biomarker validation.

The transfer of biomarkers from the discovery field to clinical use is still, despite progress, on a road filled with pitfalls. Since the emergence of proteomics, thousands of putative biomarkers have been published, often with overlapping diagnostic capacities. The strengthening of the robustness of discovery technologies, particularly in mass spectrometry, has been followed by intense discussions on establishing well-defined evaluation procedures for the identified targets to ultimately allow the clinical validation and then the clinical use of some of these biomarkers. Some of the obstacles to the evaluation process have been the lack of the availability of quick and easy-to-develop, easy-to-use, robust, specific and sensitive alternative quantitative methods when immunoaffinity-based tests are unavailable. Multiple reaction monitoring (MRM; also called selected reaction monitoring) is currently proving its capabilities as a complementary or alternative technique to ELISA for large biomarker panel evaluation. Here, we present how MRM(3) can overcome the lack of specificity and sensitivity often encountered by MRM when tracking minor proteins diluted by complex biological matrices.