Accurate Protein Quantification Research Articles

A Cole‐Cole Dielectric Relaxation Analysis of Albumin and γ‐Globulins for Protein Quantification by Electrical Impedance Spectroscopy

AbstractOptimal serum protein concentrations are vital for normal body functioning. Affordable while accurate protein quantification methods with minimum processing requirements are needed for diagnosis of related diseases. The standard automated chemistry analyzer is limited by high installation and maintenance costs. This study proposes the use of electrical impedimetric spectroscopy (EIS) as an alternative to current methods. Its practical applicability was tested using albumin and γ‐globulin or their miscellanea in three different media; water, serum and tissue‐mimicking phantoms at 25 °C. Impedance measurements were taken between frequency f=0.10 MHz to 300 MHz by an impedance analyzer. A Cole‐Cole analysis was used to elucidate the stepwise variations in the dielectric parameters of the protein medium so as to obtain empirical dielectric parameter‐protein concentration relationships and their correlation coefficients R2. From the results, linear relationships between parameters and protein concentrations with high correlation coefficients over R2=0.90 were observed. Resistance to charge transfer Rct and characteristic frequency fc were significantly altered by changing protein concentrations as compared to bulk solution resistance Rs, relaxation time constant τ and shape factor α. The relationships developed would aid in monitoring changes in body fluid protein concentrations by EIS.

Iq: an R package to estimate relative protein abundances from ion quantification in DIA-MS-based proteomics.

SummaryWe present an R package called iq to enable accurate protein quantification for label-free data-independent acquisition (DIA) mass spectrometry-based proteomics, a recently developed global approach with superior quantitative consistency. We implement the popular maximal peptide ratio extraction module of the MaxLFQ algorithm, so far only applicable to data-dependent acquisition mode using the software suite MaxQuant. Moreover, our implementation shows, for each protein separately, the validity of quantification over all samples. Hence, iq exports a state-of-the-art protein quantification algorithm to the emerging DIA mode in an open-source implementation.Availability and implementationThe open-source R package is available on CRAN, https://github.com/tvpham/iq/releases and oncoproteomics.nl/iq.Supplementary information Supplementary data are available at Bioinformatics online.

Fluorimetric-Based Method to Detect and Quantify Total S-Nitrosothiols (SNOs) in Plant Samples.

Accumulating experimental evidence indicates that S-nitrosylation (technically S-nitrosation) events have a central role in plant biology, presumably accounting for much of the widespread influence of nitric oxide (NO) on developmental, metabolic, and stress-related plant responses. Therefore, the accurate detection and quantification of S-nitrosylated proteins and peptides can be particularly useful to determine the relevance of this class of compounds in the ever-increasing number of NO-dependent signaling events described in plant systems. Up to now, the quantification of S-nitrosothiols (SNOs) in plant samples has mostly relied on the Saville reaction and the ozone-based chemiluminescence method, which lacks sensitivity and are very time-consuming, respectively. Taking advantage of the photolytic properties of S-nitrosylated proteins and peptides, the method described in this chapter allows simple, fast, and high-throughput detection of SNOs in plant samples.

Abstract 1149: Systems pathology by Optra 30 color multiplex immunofluorescence automated whole slide imaging system integrated with image analysis

Abstract Background A paradigm shift from single marker immunohistochemistry (IHC) to multiplexing in molecular histopathology has helped better understand the cellular composition and heterogeneity of sub population of cells in tissues. Presently, one needs to manually exchange filters after each multiplex scan for capturing all fluorophores per staining round. This is very cumbersome and inefficient, as this process requires rescanning the same slide multiple times. Moreover, visual assessment and quantification of biomarkers is limited by inter and intra-observer variability. We hereby present an automated multispectral whole-slide imaging system integrated with image analysis for detection of upto 30-color multiplex assay with spectral unmixing and cell segmentation functionality. Material and Methods Multiplex IHC whole slide imaging Formalin Fixed Paraffin Embedded rat brain; indirect IHC stained sections with 10 antibodies using photostable fluorophores were obtained. The staining was based on antigenic integrity and antibody binding affinity. Optra's multispectral fluorescent whole slide scanning process was optimized to image sensitive antigens in early cycles and robust antigens in latter cycles. The fully automated fluorescence scanning system imaged weakly expressing biomarkers in the visible range (480-620 nm), highly expressing targets in the violet/blue (405-440 nm), far-red (650-680 nm) and NIR (700-800 nm) range across the entire usable light spectrum (350-800 nm). ResultsWe could simultaneously detect up to 30 biomarkers within a single tissue section, using 12 staining-imaging-stripping cycles, with 10 antibodies screened per cycle. Software measures enabled separation of spectrally overlapping markers in the multiplex assay. A computer-controlled X-Y-Z stage used autofocusing at 20X/40X magnification. Integrated image analysis solution performed comprehensive evaluation of antigens of interest with accurate quantification of the target proteins based on parameters including but not limited to hue value/width, intensity threshold scoring, morphological characterization, pixel-count thresholding and color saturation. Conclusion This proposed solution is efficient compared to the current imaging methods to tackle the aforementioned image acquisition and image analysis challenges associated with multiplexing assays. Note: This abstract was not presented at the meeting. Citation Format: Abhi Gholap, Anagha P. Jadhav, Suraj Somwanshi, Gurunath Kamble, Isha Doshi, Jiaoti Huang. Systems pathology by Optra 30 color multiplex immunofluorescence automated whole slide imaging system integrated with image analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1149.

Quantitative proteomic analysis of a genetically induced prostate inflammation mouse model via custom 4-plex DiLeu isobaric labeling.

Inflammation is involved in many prostate pathologies including infection, benign prostatic hyperplasia, and prostate cancer. Preclinical models are critical to our understanding of disease mechanisms, yet few models are genetically tractable. Here, we present a comparative quantitative proteomic analysis of urine from mice with and without prostate-specific inflammation induced by conditional prostate epithelial IL-1β expression. Relative quantification and sample multiplexing was achieved using custom 4-plex N,N-dimethyl leucine (DiLeu) isobaric tags and nanoflow ultrahigh-performance liquid chromatography coupled to high-resolution tandem mass spectrometry. Each set of 4-plex DiLeu reagents allows four urine samples to be analyzed simultaneously, providing high-throughput and accurate quantification of urinary proteins. Proteins involved in the acute phase response, including haptoglobin, inter-α-trypsin inhibitor, and α1-antitrypsin 1-1, were differentially represented in the urine of mice with prostate inflammation. Mass spectrometry-based quantitative urinary proteomics represents a promising bioanalytical strategy for biomarker discovery and the elucidation of molecular mechanisms in urological research.

HOTMAQ: A Multiplexed Absolute Quantification Method for Targeted Proteomics.

Absolute quantification in targeted proteomics is challenging due to a variety of factors, including low specificity in complex backgrounds, limited analytical throughput, and wide dynamic range. To address these problems, we developed a hybrid offset-triggered multiplex absolute quantification (HOTMAQ) strategy that combines cost-effective mass difference and isobaric tags to enable simultaneous construction of an internal standard curve in the MS1 precursor scan, real-time identification of peptides at the MS2 level, and mass offset-triggered accurate quantification of target proteins in synchronous precursor selection (SPS)-MS3 spectra. This approach increases the analytical throughput of targeted quantitative proteomics by up to 12-fold. The HOTMAQ strategy was employed to verify candidate protein biomarkers in preclinical Alzheimer's disease with high accuracy. The greatly enhanced throughput and quantitative performance, paired with sample flexibility, makes HOTMAQ broadly applicable to targeted peptidomics, proteomics, and phosphoproteomics.

Quantification of Proteins Involved in Drug Metabolism and Disposition in the Human Liver Using Label-Free Global Proteomics.

There is an urgent need (recognized in FDA guidance, 2018) to optimize the dose of medicines given to patients for maximal drug efficacy and limited toxicity (precision dosing), which can be facilitated by quantitative systems pharmacology (QSP) models. Accurate quantification of proteins involved in drug clearance is essential to build and improve QSP models for any target population. Here we describe application of label-free proteomics in microsomes from 23 human livers to simultaneously quantify 188 enzymes and 66 transporters involved in xenobiotic disposition, including 17 cytochrome P450s (CYPs), 10 UDP-glucuronosyltransferases (UGTs), 7 ATP-binding cassette (ABC) transporters, and 11 solute carrier (SLC) transporters; six of these proteins are quantified for the first time. The methodology allowed quantification of thousands of proteins, allowing estimation of sample purity and understanding of global patterns of protein expression. There was overall good agreement with targeted quantification and enzyme activity data, where this was available. The effects of sex, age, genotype, and BMI on enzyme and transporter expression were assessed. Decreased expression of enzymes and transporters with increasing BMI was observed, but a tendency for older donors to have higher BMIs may have confounded this result. The effect of genotype on enzymes expression was, however, clear-cut, with CYP3A5*1/*3 genotype expressed 16-fold higher compared with its mostly inactive *3/*3 counterpart. Despite the complex, time-consuming data analysis required for label-free methodology, the advantages of the label-free method make it a valuable approach to populate a broad range of system parameters simultaneously for target patients within pharmacology and toxicology models.

Quantitative assessment of serum heat shock protein 27 for the diagnosis of epithelial ovarian cancer using targeted proteomics coupled with immunoaffinity enrichment

BackgroundHeat shock protein 27 (HSP27) may take part in the epithelial ovarian cancer (EOC) malignant process because it is elevated in the serum of EOC patients, suggesting that HSP27 may serve as an EOC biomarker to complement the standard serum carbohydrate antigen 125 (CA125) test. Thus, accurate quantification of serum HSP27 would assist the diagnosis of EOC. MethodsLiquid chromatography-tandem mass spectrometry (LC–MS/MS)-based targeted proteomics coupled with an immunoaffinity enrichment assay was developed and validated to monitor HSP27 concentrations in serum. ResultsTryptic peptide 80QLSSGVSEIR89 was selected as a surrogate analyte for quantification, and an immuno-depleted serum extract was used as a surrogate matrix. Immunoaffinity enrichment was effective for protein enrichment and sensitivity enhancement, and the resulting LOQ was 500 pg/ml (>10-fold increase). Then, serum HSP27 concentrations in EOC patients, benign ovarian tumors patients and healthy volunteers were accurately determined to be 4.95 ± 0.37 ng/ml, 2.98 ± 0.16 ng/ml and 2.82 ± 0.15 ng/ml, respectively, suggesting that the EOC samples had significantly higher concentrations of HSP27 than a sample from benign ovarian tumor patients. The experimental values for the samples were compared with those obtained from enzyme-linked immune sorbent assays (ELISAs). The ROC curve analysis showed that the combined area under the curve (AUC) for CA125 and HSP27 was 0.88, which is significantly superior to that of CA125 alone. ConclusionsTargeted proteomics coupled with immunoaffinity enrichment may provide more accurate quantification of low-abundant proteins.

Peptides releasing study of serum apolipoprotein A-I and apolipoprotein B by ID-LC-MS

Objective To investigate the digestion kinetics of Apolipoprotein A-I and B by ID-LC-MS method for accurate quantification of proteins. Methods Methodological research. The target peptides of ApoA-I and B were determined. The ApoA-I and B from 5 human serum samples on market with levels from 0.90-2.54 g/L and 0.54-1.39 g/L separately, were measured in terms of target peptides by isotope dilution liquid chromatography mass spectrometry method. The releasing amount and rate of peptides were analyzed and plotted according to different time points. The correlation coefficient R2 was calculated among peptide releasing amount between samples. Results Most peptides reached their peaks within 4 hours. The peptides VQ, DY and VS from Apo A-I, TR and FP from Apo B were released relatively slowly. After getting to their peak stage, the ratio between TEV and SIL-TEV, AK and SIL-AK, VQ and SIL-VQ presented stable state. As for Apo A-I the correlations among peptides are high, from 0.904 to 0.999. Some peptides from Apo B show lower correlations, such as TG-SV with R2 0.543 (3 h). Conclusions Peptides from Apo A-I and Apo B present different releasing properties after trypsin digestion. Proper selection of representative peptides and enzymatic conditions can benefit accurate quantification of target proteins.(Chin J Lab Med, 2018, 41: 870-874) Key words: Apolipoprotein A-I; Apolipoproteins B; Kinetics; Chromatography, liquid; Tandem mass spectrometry; Isotope labeling

Targeted Proteomics Guided by Label-free Quantitative Proteome Analysis in Saliva Reveal Transition Signatures from Health to Periodontal Disease

Periodontal diseases are among the most prevalent worldwide, but largely silent, chronic diseases. They affect the tooth-supporting tissues with multiple ramifications on life quality. Their early diagnosis is still challenging, due to lack of appropriate molecular diagnostic methods. Saliva offers a non-invasively collectable reservoir of clinically relevant biomarkers, which, if utilized efficiently, could facilitate early diagnosis and monitoring of ongoing disease. Despite several novel protein markers being recently enlisted by discovery proteomics, their routine diagnostic application is hampered by the lack of validation platforms that allow for rapid, accurate and simultaneous quantification of multiple proteins in large cohorts. Here we carried out a pipeline of two proteomic platforms; firstly, we applied open ended label-free quantitative (LFQ) proteomics for discovery in saliva (n = 67, including individuals with health, gingivitis, and periodontitis), followed by selected-reaction monitoring (SRM)-targeted proteomics for validation in an independent cohort (n = 82). The LFQ platform led to the discovery of 119 proteins with at least 2-fold significant difference between health and disease. The 65 proteins chosen for the subsequent SRM platform included 50 functionally related proteins derived from the significantly enriched processes of the LFQ data, 11 from literature-mining, and four house-keeping ones. Among those, 60 were reproducibly quantifiable proteins (92% success rate), represented by a total of 143 peptides. Machine-learning modeling led to a narrowed-down panel of five proteins of high predictive value for periodontal diseases with maximum area under the receiver operating curve >0.97 (higher in disease: Matrix metalloproteinase-9, Ras-related protein-1, Actin-related protein 2/3 complex subunit 5; lower in disease: Clusterin, Deleted in Malignant Brain Tumors 1). This panel enriches the pool of credible clinical biomarker candidates for diagnostic assay development. Yet, the quantum leap brought into the field of periodontal diagnostics by this study is the application of the biomarker discovery-through-verification pipeline, which can be used for validation in further cohorts.

Use of Cyclodextrin as a Novel Agent in the SEC-HPLC Mobile Phase to Mitigate the Interactions of Proteins or Peptide or their Impurities with the Residual Silanols of Commercial SEC-HPLC Columns with Improved Separation and Resolution.

Accurate quantification of the intact proteins, antibodies or peptides and their impurities without interaction to silanols of HPLC column. Hydroxypropyl ß Cyclodextrin (HPCD) is added in the mobile phase at different concentrations. Different commercial SEC-HPLC columns and biologics with a molecular weight ranging from 5.8kDa to 150kDa were assessed with and without cyclodextrin. Addition of non-ionic sugars such as Hydroxypropyl ß Cyclodextrin in the mobile phase, resulted improved peak performance such as theoretical plates, peak resolution, peak width, peak height, and improved quantification of aggregates in biologics such as antibodies Humira and Actemra, and peptides such as insulin. There is an increase in peak height, reduced retention time, increased plate and reduced peak width with increasing concentration of cyclodextrin studied. High ionic strength, basic amino acids such as arginine, organic solvents (with a concentration low enough not to precipitate protein), sodium perchlorate and ion pairing agents in the mobile phase used for separation of peptides, proteins and antibodies to prevent silanol interaction. These commonly used solutions are not always successful, as they not only interact with the biologic, but are sometimes, not compatible. The non-ionic cyclodextrin itself does not cause protein aggregation but prevents the nonspecific binding or interaction of protein itself and thereby allowing for improved resolution, and accurate quantification of aggregates in antibodies, and peptides. The data on the separation in presence of cyclodextrin in the mobile phase showed higher peak resolution, improved peak shape, accurate apparent molecular weight, improved efficiency, and less peak tailing for biological products. Hydroxypropyl ß Cyclodextrin in the mobile phase, resulted improved SEC-HPLC resolution, and quantitation of aggregates in biologics by preventing the interaction of biologics to silanol of the commercial SEC-HPLC columns.

SWATH mass spectrometry as a tool for quantitative profiling of the matrisome

Proteomic analysis of extracellular matrix (ECM) and ECM-associated proteins, collectively known as the matrisome, is a challenging task due to the inherent complexity and insolubility of these proteins. Here we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins in both non-enriched and ECM enriched tissue without the need for prior fractionation. Utilising a spectral library containing 201 matrisomal proteins, we compared the performance and reproducibility of SWATH MS over conventional data-dependent analysis mass spectrometry (DDA MS) in unfractionated murine lung and liver. SWATH MS conferred a 15–20% increase in reproducible peptide identification across replicate experiments in both tissue types and identified 54% more matrisomal proteins in the liver versus DDA MS. We further use SWATH MS to evaluate the quantitative changes in matrisome content that accompanies ECM enrichment. Our data shows that ECM enrichment led to a systematic increase in core matrisomal proteins but resulted in significant losses in matrisome-associated proteins including the cathepsins and proteins of the S100 family. Our proof-of-principle study demonstrates the utility of SWATH MS as a versatile tool for in-depth characterisation of the matrisome in unfractionated and non-enriched tissues. SignificanceThe matrisome is a complex network of extracellular matrix (ECM) and ECM-associated proteins that provides scaffolding function to tissues and plays important roles in the regulation of fundamental cellular processes. However, due to its inherent complexity and insolubility, proteomic studies of the matrisome typically require the application of enrichment workflows prior to MS analysis. Such enrichment strategies often lead to losses in soluble matrisome-associated components. In this study, we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins. We show that SWATH MS provides a more reproducible coverage of the matrisome compared to data-dependent analysis (DDA) MS. We also demonstrate that SWATH MS is capable of accurate quantification of matrisomal proteins without prior ECM enrichment and fractionation, which may simplify sample handling workflows and avoid losses in matrisome-associated proteins commonly linked to ECM enrichment.

Parameter estimation of stochastic models based on limited data

Progress in experimental techniques enables a more accurate quantification of genes, mRNA, and proteins at the single cell level. Provided with limited time series data from single-cell measurements, this note proposes a new quasi-Newton optimization algorithm (QNSTOP) for parameter estimation of stochastic models. To capture the stochasticity inside models and data, the random objective function is constructed based on the maximum log-likelihood of transition probabilities rather than summary statistics, which relies heavily on stochastic simulations. Simple to use and efficient, QNSTOP can find the "best" parameter vector from far away starting points in just a few iterations. Results on a bistable model match well the bistable dynamics that can only be obtained from stochastic models.

ITRAQ-Based Identification of Proteins Related to Muscle Growth in the Pacific Abalone, Haliotis discus hannai.

The abalone Haliotis discus hannai is an important aquaculture species that is grown for human consumption. However, little is known of the genetic mechanisms governing muscle growth in this species, particularly with respect to proteomics. The isobaric tag for relative and absolute quantitation (iTRAQ) method allows for sensitive and accurate protein quantification. Our study was the first to use iTRAQ-based quantitative proteomics to investigate muscle growth regulation in H. discus hannai. Among the 1904 proteins identified from six samples, 125 proteins were differentially expressed in large specimens of H. discus hannai as compared to small specimens. In the large specimens, 47 proteins were upregulated and 78 were downregulated. Many of the significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including these differentially expressed proteins, were closely related to muscle growth, including apoptosis, thyroid hormone signaling, regulation of the actin cytoskeleton, and viral myocarditis (p < 0.05). Our quantitative real-time polymerase chain reaction (qRT-PCR) analyses suggested that the alterations in expression levels observed in the differentially expressed proteins were consistent with the alterations observed in the encoding mRNAs, indicating the repeatability of our proteomic approach. Our findings contribute to the knowledge of the molecular mechanisms of muscle growth in H. discus hannai.

Isobaric Labeling-Based LC-MS/MS Strategy for Comprehensive Profiling of Human Pancreatic Tissue Proteome.

The pancreas is an organ with both endocrine and exocrine functions, and various pathologies, such as pancreatic cancer and diabetes are associated with this organ. Owing to the limited pancreatic biopsy samples available for research, it is critical to make the best use of cadaveric pancreatic tissue for biomarker studies and mechanistic understanding of pancreas-related pathologies. Discovery-phase quantitative proteomics has attracted a lot of attention for its capabilities in large-scale protein identification and accurate protein quantification. Here, we describe a workflow using isobaric labeling (tandem mass tag or TMT) based quantitative proteomics to confidently identify and quantify human pancreatic tissue proteome, including sample preparation, isobaric tag labeling, peptide level fractionation, LC-MS/MS, database search, and statistical analysis.

Towards comprehensive and quantitative proteomics for diagnosis and therapy of human disease.

Given superior analytical features, MS proteomics is well suited for the basic investigation and clinical diagnosis of human disease. Modern MS enables detailed functional characterization of the pathogenic biochemical processes, as achieved by accurate and comprehensive quantification of proteins and their regulatory chemical modifications. Here, we describe how high-accuracy MS in combination with high-resolution chromatographic separations can be leveraged to meet these analytical requirements in a mechanism-focused manner. We review the quantification methods capable of producing accurate measurements of protein abundance and posttranslational modification stoichiometries. We then discuss how experimental design and chromatographic resolution can be leveraged to achieve comprehensive functional characterization of biochemical processes in complex biological proteomes. Finally, we describe current approaches for quantitative analysis of a common functional protein modification: reversible phosphorylation. In all, current instrumentation and methods of high-resolution chromatography and MS proteomics are poised for immediate translation into improved diagnostic strategies for pediatric and adult diseases.

Accurate and Sensitive Quantification of Soy Proteins in Raw and Processed Food by Sandwich ELISA

Soy belongs to the so called 'Big 8' allergens and following food labeling directives of many different countries soy has to be labeled as ingredient if used in food. Allergen labeling statements enable sensitized patients to avoid the intake of food containing soy. The major allergenic proteins in soy are Glycinin (Gly m 6) and s-Conglycinin (Gly m 5). The sandwich ELISA RIDASCREEN®FAST Soya quantifies these two proteins in unprocessed as well as in processed food. The following performance characteristics were validated: Selectivity, limit of detection, limit of quantification, precision, recovery, and robustness. First results from proficiency test rounds verified these validation results. In summary, the assay shows high precision and very good recoveries for all different types of samples. It is robust against normal occurring variation within a laboratory. Compared to other test-kits on the market, this ELISA is suitable for the recognition of unheated to strongly heated soy proteins in various foods. Therefore the test can be used for most samples on the market that contain intact soy protein.

Elemental Mass Spectrometry for Absolute Intact Protein Quantification without Protein-Specific Standards: Application to Snake Venomics.

Absolute protein quantification methods based on molecular mass spectrometry usually require stable isotope-labeled analogous standards for each target protein or peptide under study, which in turn must be certified using natural standards. In this work, we report a direct and accurate methodology based on capLC-ICP-QQQ and online isotope dilution analysis for the absolute and sensitive quantification of intact proteins. The combination of the postcolumn addition of 34S and a generic S-containing internal standard spiked to the sample provides full compound independent detector response and thus protein quantification without the need for specific standards. Quantitative recoveries, using a chromatographic core-shell C4 column for the various protein species assayed were obtained (96-100%). Thus, the proposed strategy enables the accurate quantification of proteins even if no specific standards are available for them. In addition, to the best of our knowledge, we obtained the lowest detection limits reported in the quantitative analysis of intact proteins by direct measurement of sulfur with ICPMS (358 fmol) and protein (ranging from 7 to 15 fmol depending on the assayed protein). The quantitative results for individual and simple mixtures of model proteins were statistically indistinguishable from the manufacturer's values. Finally, the suitability of the strategy for real sample analysis (including quantitative protein recovery from the column) was illustrated for the individual absolute quantification of the proteins and whole protein content in a venom sample. Parallel capLC-ESI-QTOF analysis was employed to identify the proteins, a prerequisite to translate the mass of quantified S for each chromatographic peak into individual protein mass.

Systems biology-opportunities and challenges: the application of proteomics to study the cardiovascular extracellular matrix.

Systems biology approaches including proteomics are becoming more widely used in cardiovascular research. In this review article, we focus on the application of proteomics to the cardiac extracellular matrix (ECM). ECM remodelling is a hallmark of many cardiovascular diseases. Proteomic techniques using mass spectrometry (MS) provide a platform for the comprehensive analysis of ECM proteins without a priori assumptions. Proteomics overcomes various constraints inherent to conventional antibody detection. On the other hand, studies that use whole tissue lysates for proteomic analysis mask the identification of the less abundant ECM constituents. In this review, we first discuss decellularization-based methods that enrich for ECM proteins in cardiac tissue, and how targeted MS allows for accurate protein quantification. The second part of the review will focus on post-translational modifications including hydroxylation and glycosylation and on the release of matrix fragments with biological activity (matrikines), all of which can be interrogated by proteomic techniques.

Preparation of trypsin aptamer modified silica particles by surface initiated atom transfer radical polymerization for proteome identification

Highly efficient and complete protein digestion is vital for achieving accurate protein quantification.