Deep quantitative proteomics of North American Pacific coast star tunicate (Botryllus schlosseri).

Botryllus schlosseri, is a model marine invertebrate for studying immunity, regeneration, and stress-induced evolution. Conditions for validating its predicted proteome were optimized using nanoElute® 2 deep-coverage LCMS, revealing up to 4930 protein groups and 20,984 unique peptides per sample. Spectral libraries were generated and filtered to remove interferences, low-quality transitions, and only retain proteins with >3 unique peptides. The resulting DIA assay library enabled label-free quantitation of 3426 protein groups represented by 22,593 unique peptides. Quantitative comparisons of single systems from a laboratory-raised with two field-collected populations revealed (1) a more unique proteome in the laboratory-raised population, and (2) proteins with high/low individual variabilities in each population. DNA repair/replication, ion transport, and intracellular signaling processes were distinct in laboratory-cultured colonies. Spliceosome and Wnt signaling proteins were the least variable (highly functionally constrained) in all populations. In conclusion, we present the first colonial tunicate's deep quantitative proteome analysis, identifying functional protein clusters associated with laboratory conditions, different habitats, and strong versus relaxed abundance constraints. These results empower research on B. schlosseri with proteomics resources and enable quantitative molecular phenotyping of changes associated with transfer from in situ to ex situ and from in vivo to in vitro culture conditions.

Deciphering a proteomic signature for the early detection of breast cancer from breast milk: the role of quantitative proteomics.

INTRODUCTION: Breast cancer is one of the most prevalent cancers among women in the United States. Current research regarding breast milk has been focused on the composition and its role in infant growth and development. There is little information about the proteins, immune cells, and epithelial cells present in breast milk which can be indicative of the emergence of BC cells and tumors. AREAS COVERED: We summarize all breast milk studies previously done in our group using proteomics. These studies include 1D-PAGE and 2D-PAGE analysis of breast milk samples, which include within woman and across woman comparisons to identify dysregulated proteins in breast milk and the roles of these proteins in both the development of BC and its diagnosis. Our projected outlook for the use of milk for cancer detection is also discussed. EXPERT OPINION: Analyzing the samples by multiple methods allows one to interrogate a set of samples with various biochemical methods that complement each other, thus providing a more comprehensive proteome. Complementing methods like 1D-PAGE, 2D-PAGE, in-solution digestion and proteomics analysis with PTM-omics, peptidomics, degradomics, or interactomics will provide a better understanding of the dysregulated proteins, but also the modifications or interactions between these proteins.

Absolute quantitation of human wild-type DNAI1 protein in lung tissue using a nanoLC-PRM-MS-based targeted proteomics approach coupled with immunoprecipitation.

BACKGROUND: Dynein axonemal intermediate chain 1 protein (DNAI1) plays an essential role in cilia structure and function, while its mutations lead to primary ciliary dyskinesia (PCD). Accurate quantitation of DNAI1 in lung tissue is crucial for comprehensive understanding of its involvement in PCD, as well as for developing the potential PCD therapies. However, the current protein quantitation method is not sensitive enough to detect the endogenous level of DNAI1 in complex biological matrix such as lung tissue. METHODS: In this study, a quantitative method combining immunoprecipitation with nanoLC-MS/MS was developed to measure the expression level of human wild-type (WT) DNAI1 protein in lung tissue. To our understanding, it is the first immunoprecipitation (IP)-MS based method for absolute quantitation of DNAI1 protein in lung tissue. The DNAI1 quantitation was achieved through constructing a standard curve with recombinant human WT DNAI1 protein spiked into lung tissue matrix. RESULTS: This method was qualified with high sensitivity and accuracy. The lower limit of quantitation of human DNAI1 was 4 pg/mg tissue. This assay was successfully applied to determine the endogenous level of WT DNAI1 in human lung tissue. CONCLUSIONS: The results clearly demonstrate that the developed assay can accurately quantitate low-abundance WT DNAI1 protein in human lung tissue with high sensitivity, indicating its high potential use in the drug development for DNAI1 mutation-caused PCD therapy.

Normics: Proteomic Normalization by Variance and Data-Inherent Correlation Structure.

Several algorithms for the normalization of proteomic data are currently available, each based on a priori assumptions. Among these is the extent to which differential expression (DE) can be present in the dataset. This factor is usually unknown in explorative biomarker screens. Simultaneously, the increasing depth of proteomic analyses often requires the selection of subsets with a high probability of being DE to obtain meaningful results in downstream bioinformatical analyses. Based on the relationship of technical variation and (true) biological DE of an unknown share of proteins, we propose the "Normics" algorithm: Proteins are ranked based on their expression level-corrected variance and the mean correlation with all other proteins. The latter serves as a novel indicator of the non-DE likelihood of a protein in a given dataset. Subsequent normalization is based on a subset of non-DE proteins only. No a priori information such as batch, clinical, or replicate group is necessary. Simulation data demonstrated robust and superior performance across a wide range of stochastically chosen parameters. Five publicly available spike-in and biologically variant datasets were reliably and quantitively accurately normalized by Normics with improved performance compared to standard variance stabilization as well as median, quantile, and LOESS normalizations. In complex biological datasets Normics correctly determined proteins as being DE that had been cross-validated by an independent transcriptome analysis of the same samples. In both complex datasets Normics identified the most DE proteins. We demonstrate that combining variance analysis and data-inherent correlation structure to identify non-DE proteins improves data normalization. Standard normalization algorithms can be consolidated against high shares of (one-sided) biological regulation. The statistical power of downstream analyses can be increased by focusing on Normics-selected subsets of high DE likelihood.

IQMMA: Efficient MS1 Intensity Extraction Pipeline Using Multiple Feature Detection Algorithms for DDA Proteomics.

One of the key steps in data dependent acquisition (DDA) proteomics is detection of peptide isotopic clusters, also called "features", in MS1 spectra and matching them to MS/MS-based peptide identifications. A number of peptide feature detection tools became available in recent years, each relying on its own matching algorithm. Here, we provide an integrated solution, the intensity-based Quantitative Mix and Match Approach (IQMMA), which integrates a number of untargeted peptide feature detection algorithms and returns the most probable intensity values for the MS/MS-based identifications. IQMMA was tested using available proteomic data acquired for both well-characterized (ground truth) and real-world biological samples, including a mix of Yeast and E. coli digests spiked at different concentrations into the Human K562 digest used as a background, and a set of glioblastoma cell lines. Three open-source feature detection algorithms were integrated: Dinosaur, biosaur2, and OpenMS FeatureFinder. None of them was found optimal when applied individually to all the data sets employed in this work; however, their combined use in IQMMA improved efficiency of subsequent protein quantitation. The software implementing IQMMA is freely available at https://github.com/PostoenkoVI/IQMMA under Apache 2.0 license.

Bioconjugation of Meldrum's acid activated furan: A detergent compatible assay for protein quantitation.

A simple yet efficient assay for the quantitation of proteins ranging from plasma proteins to purified proteins from whole cell lysate, based on the bioconjugation reaction between protein and Meldrum's acid Activated Furan (MAF) is described. This easy to use, sensitive method is based on the conjugation of amine functionalities present on the protein with MAF to form the corresponding Donor Acceptor Stenhouse Adducts (DASAs) with characteristic absorption in the visible region. The reaction is rapid as well as reproducible and shows a proportionate increase in color change over a broad range of protein concentration. The assay was found to be sensitive up to 0.125 mg/mL concentration of the protein and was compatible with most of the commonly employed detergents and isolation protocols which makes it ideal for the estimation of protein samples containing detergents. Another striking feature of this protocol is its tolerance towards other major interference contributors such as chelating agents, reducing agents, carbohydrates and protease inhibitors.

Recent advances in isobaric labeling and applications in quantitative proteomics.

Mass spectrometry (MS) has emerged at the forefront of quantitative proteomic techniques. Liquid chromatography-mass spectrometry (LC-MS) can be used to determine abundances of proteins and peptides in complex biological samples. Several methods have been developed and adapted for accurate quantification based on chemical isotopic labeling. Among various chemical isotopic labeling techniques, isobaric tagging approaches rely on the analysis of peptides from MS2-based quantification rather than MS1-based quantification. In this review, we will provide an overview of several isobaric tags along with some recent developments including complementary ion tags, improvements in sensitive quantitation of analytes with lower abundance, strategies to increase multiplexing capabilities, and targeted analysis strategies. We will also discuss limitations of isobaric tags and approaches to alleviate these restrictions through bioinformatic tools and data acquisition methods. This review will highlight several applications of isobaric tags, including biomarker discovery and validation, thermal proteome profiling, cross-linking for structural investigations, single-cell analysis, top-down proteomics, along with applications to different molecules including neuropeptides, glycans, metabolites, and lipids, while providing considerations and evaluations to each application.

Quantitative mass spectrometry-based analysis of proteins related to cattle and their products - Focus on cows' milk beta-casein proteoforms.

Modern mass spectrometers can accurately measure thousands of compounds in complex mixtures over a given liquid chromatograph method, depending on desired outcome and method duration. This stream of analytical chemistry has wide ranging application across food, pharma, environmental, forensics, clinical and research. With consistent pressure on both the ruminant production and product industries to face new and substantial challenges, liquid chromatography-mass spectrometry (LC-MS) is an ideal tool to identify, detect and quantify markers of breeding, production and adaption to support both research and industry to overcome these challenges. Herein, we provide a description of the theoretical basis and framework for LC-MS as a rapidly developing technique and highlight its application in measuring cattle and cattle product traits through protein quantitation with specific focus on beta-casein proteoforms.

Intact quantitative bioanalytical method development and fit-for-purpose validation of a monoclonal antibody and its related fab fragment in human vitreous and aqueous humor using LC-HRMS.

Ranibizumab is an FDA-approved drug used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, macular edema, and myopic choroidal neovascularization. Bevacizumab is another drug often used off-label to treat wet AMD. In order to reduce unwanted angiogenesis, ranibizumab and bevacizumab target circulating VEGF-A in the eye. Concentration levels in human vitreous and aqueous humor can be used to provide valuable efficacy information. However, vitreous and aqueous humor's aqueous environment, and vitreous humor's viscosity, as well as the stickiness of the analytes can provide bioanalytical challenges. In this manuscript, we describe the development, optimization, and fit-for-purpose validation of an LC-HRMS method designed for intact quantitative bioanalysis of ranibizumab and bevacizumab in human vitreous and aqueous humor following intravitreal administration. In order to fully develop this method, evaluations were conducted to optimize the conditions, including the data processing model (extracted ion chromatograms (XICs) vs deconvolution), carryover mitigation, sample preparation scheme optimization for surrogate and primary matrices, use of internal standard/immunocapture/deglycosylation, and optimization of the extraction and dilution procedure, as well as optimization of the liquid chromatography and mass spectrometry conditions. Once the method was fully optimized, a fit-for-purpose validation was conducted, including matrix parallelism, with a linear calibration range of 10 to 200 µg/mL. The development of this intact quantitative method using LC-HRMS provides a proof-of-concept template for challenging, but valuable new and exciting bioanalytical techniques.

Hevea brasiliensis latex proteomics: a review of analytical methods and the way forward.

Natural rubber or latex from the Hevea brasiliensis is an important commodity in various economic sectors in today's modern society. Proteins have been detected in latex since the early twentieth century, and they are known to regulate various biological pathways within the H. brasiliensis trees such as the natural rubber biosynthesis, defence against pathogens, wound healing, and stress tolerance. However, the exact mechanisms of the pathways are still not clear. Proteomic analyses on latex have found various proteins and revealed how they fit into the mechanisms of the biological pathways. In the past three decades, there has been rapid latex protein identification due to the improvement of latex protein extraction methods, as well as the emergence of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). In this manuscript, we reviewed the methods of latex protein extraction that keeps on improving over the past three decades as well as the results of numerous latex protein identification and quantitation.

Development and fit-for-purpose verification of an LC-MS method for quantitation of hemagglutinin and neuraminidase proteins in influenza virus-like particle vaccine candidates.

Recombinant influenza Virus-Like Particle (VLP) vaccines are promising vaccine candidates to prevent influenza, contain two major viral antigenic glycoproteins, Hemagglutinin (HA) and Neuraminidase (NA), on the surface of recombinant VLPs. Accurate quantitation of the mass of these antigenic proteins is important to ensure the product quality and proper dosing. Currently, Single Radial Immunodiffusion (SRID) is a recognized assay for determination of the HA immuno-reactive concentration (potency) in vaccine products, based on immuno-reactivity of HA with strain-specific antisera. The SRID assay, however, requires availability of strain-specific and properly calibrated reagents, which can be time-consuming to generate and calibrate. In addition, the assay is not suitable for quantitation of low abundant proteins, such as NA. In order to accelerate the overall production cycle, we have developed and optimized a high-resolution (HR) LC-MS method for absolute quantitation of both HA and NA protein concentrations in influenza VLP vaccine candidates. In this work, we present the method development, optimization and verification of its suitability for the intended purpose, as a prerequisite for its potential application in Quality Control, by assessing specificity, precision and accuracy, detection characteristics, and dynamic linear range. The method can be also used for other HA/NA containing preparations including in-process samples, purified proteins, whole virus preparations, nano-particle and egg-based vaccine preparations, or for calibration of SRID reference antigens.

LC/MS/MS-based quantitation of pig and human S100A1 protein in cardiac tissues: Application to gene therapy.

The S100A1 protein is a target of interest for the treatment of heart failure as it has been previously reported to be depleted in failing cardiomyocytes. A gene therapy approach leading to increased expression levels of the protein directly in the heart could potentially lead to restoration of contractile function and improve overall cell survival. S100A1 is a relatively small soluble protein that is extremely well conserved across species with only a single amino acid difference between the sequences in human and pig, a commonly used pre-clinical model for evaluation of efficacy, biodistribution and safety for cardiac-directed gene therapy approaches. This high homology presents a bioanalytical challenge for the accurate detection and quantitation of both endogenous (pig) and exogenous (human) transduced S100A1 proteins post treatment using a human S100A1 gene therapy in pigs. Here we present a sensitive and selective LC-MS/MS approach that can easily differentiate and simultaneously quantitate both human and pig S100A1 proteins. Additionally, we report on a detailed profiling of S100A1 protein in various pig tissues, a comprehensive evaluation of S100A1 distribution in pig hearts and a comparison to S100A1 levels in human cardiac samples.

Functional analysis of synovial fluid from osteoarthritic knee and carpometacarpal joints unravels different molecular profiles.

OBJECTIVE: In this work, we aimed to elucidate the molecular mechanisms driving primary OA. By studying the dynamics of protein expression in two different types of OA joints we searched for similarities and disparities to identify key molecular mechanisms driving OA. METHODS: For this purpose, human SF samples were obtained from CMC-I OA and knee joint of OA patients. SF samples were analysed by label-free quantitative liquid chromatography mass spectrometry. Disease-relevant proteins identified in proteomics studies, such as clusterin, paraoxonase/arylesterase 1 (PON1) and transthyretin were validated by enzyme-linked immunosorbent assays, and on the mRNA level by droplet digital PCR. Functional studies were performed in vitro using primary chondrocytes. RESULTS: Differential proteomic changes were observed in the concentration of 40 proteins including clusterin, PON1 and transthyretin. Immunoassay analyses of clusterin, PON1, transthyretin and other inflammatory cytokines confirmed significant differences in protein concentration in SF of CMC-I and knee OA patients, with primarily lower protein expression levels in CMC-I. Functional studies on chondrocytes unequivocally demonstrated that stimulation with SF obtained from knee OA, in contrast to CMC-I OA joint, caused a significant upregulation in pro-inflammatory response, cell death and hypertrophy. CONCLUSION: This study demonstrates that differential expression of molecular players in SF from different OA joints evokes diverse effects on primary chondrocytes. The pathomolecular mechanisms of OA may significantly differ in various joints, a finding that brings a new dimension into the pathogenesis of primary OA.

Validation of a multiplexed LC-MS/MS clinical assay to quantify insulin-like growth factor-binding proteins in human serum and its application in a clinical study.

Circulating insulin-like growth factor-binding proteins (IGFBPs) continue to gain attention as biomarkers of drug activities on insulin like growth factor (IGF)/IGF receptor signaling pathways. A multiplexed LC-MS/MS method was validated for the absolute quantitation of IGFBPs in human serum. The method was used to measure screening concentrations of IGFBPs in spinal and bulbar muscular atrophy (SBMA) patients in a phase 2 clinical trial. Concentrations of IGFBP 1, 2, 3, and 5 were simultaneously determined based on representative signature peptides derived from an optimized trypsin digestion procedure. Signature peptide levels were absolutely quantitated using a sensitive/specific targeted LC-MS/MS method. Corresponding mass-shifted, stable isotope-labeled peptides were employed as internal standards. A true blank matrix for the quantitation of IGFBPs was not available since they are endogenous proteins in human serum. In this method, calibration standards/curves were prepared using authentic synthetic peptides spiked into a surrogate matrix. The surrogate matrix was generated from human serum treated in the same way as the study samples, but using iodoacetic acid instead of iodoacetamide as the alkylation reagent. This surrogate matrix approach allowed for the direct and sensitive/specific quantification of IGFBP 1, 2, 3, and 5 due to the lack of any endogenous background. Equivalent matrix effect and recovery of analytes was achieved for the authentic and surrogate matrices. The fully validated LC-MS/MS assay will allow further evaluation of the utility of IGFBP biomarkers in clinical trials.

Multiple Reaction Monitoring for the Quantitation of Serum Protein Glycosylation Profiles: Application to Ovarian Cancer.

Protein glycosylation fingerprints are widely recognized as potential markers for disease states, and indeed differential glycosylation has been identified in multiple types of autoimmune diseases and several types of cancer. However, releasing the glycans leave the glycoproteins unknown; therefore, there exists a need for high-throughput methods that allow quantification of site- and protein-specific glycosylation patterns from complex biological mixtures. In this study, a targeted multiple reaction monitoring (MRM)-based method for the protein- and site-specific quantitation involving serum proteins immunoglobulins A, G and M, alpha-1-antitrypsin, transferrin, alpha-2-macroglobulin, haptoglobin, alpha-1-acid glycoprotein and complement C3 was developed. The method is based on tryptic digestion of serum glycoproteins, followed by immediate reverse phase UPLC-QQQ-MS analysis of glycopeptides. To quantitate protein glycosylation independent of the protein serum concentration, a nonglycosylated peptide was also monitored. Using this strategy, 178 glycopeptides and 18 peptides from serum glycoproteins are analyzed with good repeatability (interday CVs of 3.65-21-92%) in a single 17 min run. To assess the potential of the method, protein glycosylation was analyzed in serum samples from ovarian cancer patients and controls. A training set consisting of 40 cases and 40 controls was analyzed, and differential analyses were performed to identify aberrant glycopeptide levels. All findings were validated in an independent test set (n = 44 cases and n = 44 controls). In addition to the differential glycosylation on the immunoglobulins, which was reported previously, aberrant glycosylation was also observed on each of the glycoproteins, which could be corroborated in the test set. This report shows the development of a method for targeted protein- and site-specific glycosylation analysis and the potential of such methods in biomarker development.

Evaluation of different peptide fragmentation types and mass analyzers in data-dependent methods using an Orbitrap Fusion Lumos Tribrid mass spectrometer.

One of the major additions in MS technology has been the irruption of the Orbitrap mass analyzer, which has boosted the proteomics analyses of biological complex samples since its introduction. Here, we took advantage of the capabilities of the new Orbitrap Fusion Lumos Tribrid mass spectrometer to assess the performance of different data-dependent acquisition methods for the identification and quantitation of peptides and phosphopeptides in single-shot analysis of human whole cell lysates. Our study explored the capabilities of tri-hibrid mass spectrometers for (phospho-) peptide identification and quantitation using different gradient lengths, sample amounts, and combinations of different peptide fragmentation types and mass analyzers. Moreover, the acquisition of the same complex sample with different acquisition methods resulted in the generation of a dataset to be used as a reference for further analyses, and a starting point for future optimizations in particular applications.

Determination of Rate-Limiting Factor for Formation of Beta-Catenin Destruction Complexes Using Absolute Protein Quantification.

Wnt/ß-catenin signaling plays important roles in both ontogenesis and development. In the absence of a Wnt stimulus, ß-catenin is degraded by a multiprotein "destruction complex" that includes Axin, APC, GSK3B, and FBXW11. Although the key molecules required for transducing Wnt signals have been identified, a quantitative understanding of this pathway has been lacking. Here, we calculated the absolute number of ß-catenin destruction complexes by absolute protein quantification using LC-MS/MS. Similar amounts of destruction complex-constituting proteins and ß-catenin interacted, and the number of destruction complexes was calculated to be about 1468 molecules/cell. We demonstrated that the calculated number of destruction complexes was valid for control of the ß-catenin destruction rate under steady-state conditions. Interestingly, APC had the minimum expression level among the destruction complex components at about 2233 molecules/cell, and this number approximately corresponded to the calculated number of destruction complexes. Decreased APC expression by siRNA transfection decreased the number of destruction complexes, resulting in ß-catenin accumulation and stimulation of the transcriptional activity of T-cell factor. Taken together, our results suggest that the amount of APC expression is the rate-limiting factor for the constitution of ß-catenin destruction complexes.

Quantitation of 87 Proteins by nLC-MRM/MS in Human Plasma: Workflow for Large-Scale Analysis of Biobank Samples.

A multiple reaction monitoring (MRM) assay was developed for precise quantitation of 87 plasma proteins including the three isoforms of apolipoprotein E (APOE) associated with cardiovascular diseases using nanoscale liquid chromatography separation and stable isotope dilution strategy. The analytical performance of the assay was evaluated and we found an average technical variation of 4.7% in 4-5 orders of magnitude dynamic range (≈0.2 mg/L to 4.5 g/L) from whole plasma digest. Here, we report a complete workflow, including sample processing adapted to 96-well plate format and normalization strategy for large-scale studies. To further investigate the MS-based quantitation the amount of six selected proteins was measured by routinely used clinical chemistry assays as well and the two methods showed excellent correlation with high significance (p-value < 10e-5) for the six proteins, in addition for the cardiovascular predictor factor, APOB: APOA1 ratio (r = 0.969, p-value < 10e-5). Moreover, we utilized the developed assay for screening of biobank samples from patients with myocardial infarction and performed the comparative analysis of patient groups with STEMI (ST- segment elevation myocardial infarction), NSTEMI (non ST- segment elevation myocardial infarction) and type-2 AMI (type-2 myocardial infarction) patients.

Selective Reaction Monitoring Mass Spectrometry for Quantitation of Glycolytic Enzymes in Postmortem Brain Samples.

Patients with psychiatric disorders exhibit dysfunctions in peripheral and central metabolism. This may be a root cause of impaired neuronal function, manifested as changes in mood, behavior, and cognitive capabilities in patients suffering with these conditions. Here we describe a selective reaction monitoring mass spectrometry (SRM-MS)-based targeted proteomic protocol for precise simultaneous quantitation of three glycolytic enzymes in postmortem brain tissue extracts. The SRM-MS approach has several advantages in terms of sensitivity, reproducibility, and reduced sample consumption, compared to traditional MS methods.