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.

Mitotic deacetylase complex (MiDAC) recognizes the HIV-1 core promoter to control activated viral gene expression.

The human immunodeficiency virus (HIV) integrates into the host genome forming latent cellular reservoirs that are an obstacle for cure or remission strategies. Viral transcription is the first step in the control of latency and depends upon the hijacking of the host cell RNA polymerase II (Pol II) machinery by the 5' HIV LTR. Consequently, "block and lock" or "shock and kill" strategies for an HIV cure depend upon a full understanding of HIV transcriptional control.

Development of an Untargeted LC-MS Metabolomics Method with Postcolumn Infusion for Matrix Effect Monitoring in Plasma and Feces.

Untargeted metabolomics based on reverse phase LC-MS (RPLC-MS) plays a crucial role in biomarker discovery across physiological and disease states. Standardizing the development process of untargeted methods requires paying attention to critical factors that are under discussed or easily overlooked, such as injection parameters, performance assessment, and matrix effect evaluation. In this study, we developed an untargeted metabolomics method for plasma and fecal samples with the optimization and evaluation of these factors.

Substantial downregulation of mitochondrial and peroxisomal proteins during acute kidney injury revealed by data-independent acquisition proteomics.

Acute kidney injury (AKI) manifests as a major health concern, particularly for the elderly. Understanding AKI-related proteome changes is critical for prevention and development of novel therapeutics to recover kidney function and to mitigate the susceptibility for recurrent AKI or development of chronic kidney disease. In this study, mouse kidneys were subjected to ischemia-reperfusion injury, and the contralateral kidneys remained uninjured to enable comparison and assess injury-induced changes in the kidney proteome.

Localization and Quantification of Post-Translational Modifications of Proteins Using Electron Activated Dissociation Fragmentation on a Fast-Acquisition Time-of-Flight Mass Spectrometer.

Protein post-translational modifications (PTMs) are crucial and dynamic players in a large variety of cellular processes and signaling. Proteomic technologies have emerged as the method of choice to profile PTMs. However, these analyses remain challenging due to potential low PTM stoichiometry, the presence of multiple PTMs per proteolytic peptide, PTM site localization of isobaric peptides, and neutral losses.

Accelerated Protein Biomarker Discovery from FFPE Tissue Samples Using Single-Shot, Short Gradient Microflow SWATH MS.

We reported and evaluated a microflow, single-shot, short gradient SWATH MS method intended to accelerate the discovery and verification of protein biomarkers in preclassified clinical specimens. The method uses a 15 min gradient microflow-LC peptide separation, an optimized SWATH MS window configuration, and OpenSWATH software for data analysis. We applied the method to a cohort containing 204 FFPE tissue samples from 58 prostate cancer patients and 10 benign prostatic hyperplasia patients.

Mapping Citrullinated Sites in Multiple Organs of Mice Using Hypercitrullinated Library.

Protein citrullination (or deimination), an irreversible post-translational modification, has been implicated in several physiological and pathological processes, including gene expression regulation, apoptosis, rheumatoid arthritis, and Alzheimer's disease. Several research studies have been carried out on citrullination under many conditions. However, until now, challenges in sample preparation and data analysis have made it difficult to confidently identify a citrullinated protein and assign the citrullinated site.

Highly Reproducible Automated Proteomics Sample Preparation Workflow for Quantitative Mass Spectrometry.

Sample preparation for protein quantification by mass spectrometry requires multiple processing steps including denaturation, reduction, alkylation, protease digestion, and peptide cleanup. Scaling these procedures for the analysis of numerous complex biological samples can be tedious and time-consuming, as there are many liquid transfer steps and timed reactions where technical variations can be introduced and propagated. We established an automated sample preparation workflow with a total processing time for 96 samples of 5 h, including a 2 h incubation with trypsin.

Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry.

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale.

Statistical control of peptide and protein error rates in large-scale targeted data-independent acquisition analyses.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the main method for high-throughput identification and quantification of peptides and inferred proteins. Within this field, data-independent acquisition (DIA) combined with peptide-centric scoring, as exemplified by the technique SWATH-MS, has emerged as a scalable method to achieve deep and consistent proteome coverage across large-scale data sets. We demonstrate that statistical concepts developed for discovery proteomics based on spectrum-centric scoring can be adapted to large-scale DIA experiments that have been analyzed with peptide-centric scoring strategies, and we provide guidance on their application.

Generation of High-Quality SWATH Acquisition Data for Label-free Quantitative Proteomics Studies Using TripleTOF Mass Spectrometers.

Data-independent acquisition is a powerful mass spectrometry technique that enables comprehensive MS and MS/MS analysis of all detectable species, providing an information rich data file that can be mined deeply. Here, we describe how to acquire high-quality SWATH Acquisition data to be used for large quantitative proteomic studies. We specifically focus on using variable sized Q1 windows for acquisition of MS/MS data for generating higher specificity quantitative data.

Multiplexed, Scheduled, High-Resolution Parallel Reaction Monitoring on a Full Scan QqTOF Instrument with Integrated Data-Dependent and Targeted Mass Spectrometric Workflows.

Recent advances in commercial mass spectrometers with higher resolving power and faster scanning capabilities have expanded their functionality beyond traditional data-dependent acquisition (DDA) to targeted proteomics with higher precision and multiplexing. Using an orthogonal quadrupole time-of flight (QqTOF) LC-MS system, we investigated the feasibility of implementing large-scale targeted quantitative assays using scheduled, high resolution multiple reaction monitoring (sMRM-HR), also referred to as parallel reaction monitoring (sPRM). We assessed the selectivity and reproducibility of PRM, also referred to as parallel reaction monitoring, by measuring standard peptide concentration curves and system suitability assays.

Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) Analysis for Characterization and Quantification of Histone Post-translational Modifications.

Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. High throughput characterization of histone PTMs is mostly performed by using nano-liquid chromatography coupled to mass spectrometry. However, limitations in speed and stochastic sampling of data dependent acquisition methods in MS lead to incomplete discrimination of isobaric peptides and loss of low abundant species.

Engineered metalloregulation of azide binding affinity and reduction potential of horse heart myoglobin.

Metal ion binding to a previously reported variant of horse heart myoglobin (Lys45Glu/Lys63Glu) with a metal ion binding site on the surface of the protein that is adjacent to the haem binding site has been shown to influence ligand binding and electrochemical properties of the protein. For example, the K(d) (μM) for binding of azide to this variant decreases from 277 ± 9 to 32 ± 3 following addition of a saturating concentration of Mn(2+) (the value for the wild-type protein under the same conditions is 26 ± 1). Similarly, the midpoint reduction potential E(m) (mV vs.

Recommendations for mass spectrometry data quality metrics for open access data (corollary to the Amsterdam principles).

Policies supporting the rapid and open sharing of proteomic data are being implemented by the leading journals in the field. The proteomics community is taking steps to ensure that data are made publicly accessible and are of high quality, a challenging task that requires the development and deployment of methods for measuring and documenting data quality metrics. On September 18, 2010, the U.

Recommendations for mass spectrometry data quality metrics for open access data (corollary to the Amsterdam principles).

Policies supporting the rapid and open sharing of proteomic data are being implemented by the leading journals in the field. The proteomics community is taking steps to ensure that data are made publicly accessible and are of high quality, a challenging task that requires the development and deployment of methods for measuring and documenting data quality metrics. On September 18, 2010, the U.

Recommendations for mass spectrometry data quality metrics for open access data (corollary to the Amsterdam Principles).

Policies supporting the rapid and open sharing of proteomic data are being implemented by the leading journals in the field. The proteomics community is taking steps to ensure that data are made publicly accessible and are of high quality, a challenging task that requires the development and deployment of methods for measuring and documenting data quality metrics. On September 18, 2010, the U.

Recommendations for mass spectrometry data quality metrics for open access data (corollary to the Amsterdam Principles).

Policies supporting the rapid and open sharing of proteomic data are being implemented by the leading journals in the field. The proteomics community is taking steps to ensure that data are made publicly accessible and are of high quality, a challenging task that requires the development and deployment of methods for measuring and documenting data quality metrics. On September 18, 2010, the United States National Cancer Institute convened the "International Workshop on Proteomic Data Quality Metrics" in Sydney, Australia, to identify and address issues facing the development and use of such methods for open access proteomics data.

CysTRAQ - A combination of iTRAQ and enrichment of cysteinyl peptides for uncovering and quantifying hidden proteomes.

Shotgun proteomics is capable of characterizing differences in both protein quality and quantity, and has been applied in various biomedical applications. Unfortunately, the high complexity and dynamic range of proteins in studied samples, clinical in particular, often hinders the identification of relevant proteins. Indeed, information-rich, low abundance proteins often remain undetected, whereas repeatedly reported altered concentrations in high abundance proteins are often ambiguous and insignificant.

A novel strategy for the targeted analysis of protein and peptide metabolites.

In many biological applications such as epitope discovery or drug metabolism studies, the detection of naturally processed exogenous proteins (e.g. vaccines or peptide therapeutics) and their metabolites is frequently complicated by the presence of a complex endogenous mixture of closely related or even identical compounds.

Quantitative protein determination for CYP induction via LC-MS/MS.

The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations.

A comparison of MS/MS-based, stable-isotope-labeled, quantitation performance on ESI-quadrupole TOF and MALDI-TOF/TOF mass spectrometers.

The peptide-based quantitation accuracy and precision of LC-ESI (QSTAR Elite) and LC-MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ-labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.