Custom Workflow for the Confident Identification of Sulfotyrosine-Containing Peptides and Their Discrimination from Phosphopeptides.

Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalyzed by Golgi-resident tyrosyl protein sulfo transferases (TPSTs). Information on sY in humans is currently limited to ∼50 proteins, with only a handful having verified sites of sulfation. As such, the contribution of sulfation to the regulation of biological processes remains poorly defined.

Serum protein signatures differentiate paediatric autoimmune/inflammatory disorders.

Because of their rarity, limited awareness among non-specialists, and significant overlaps in their clinical presentation, childhood autoimmune/inflammatory conditions represent a diagnostic and therapeutic challenge. Juvenile idiopathic arthritis (JIA), with its 7 sub-forms, is the most common paediatric "rheumatic" disease. Juvenile-onset systemic lupus erythematosus (jSLE) is a severe autoimmune/inflammatory disease that can affect any organ system and shares clinical features with JIA.

A Hybrid Virtual Fracture Clinic is Safe and Efficacious in the COVID-19 Era: Stay at Home and Save Lives.

Introduction The coronavirus disease 2019 (COVID-19) pandemic necessitated a change in the manner outpatient fracture clinics are conducted due to the need to reduce footfall in hospitals. While studies regarding virtual fracture clinics have shown these to be useful and effective, they focus exclusively on remote consultations. However, our service was bespoke to the patient - either a face-to-face, a telephone consultation or both, depending on patient need - a 'hybrid virtual fracture clinic' (HVFC).

An OMICs-based meta-analysis to support infection state stratification.

Motivation: A fundamental problem for disease treatment is that while antibiotics are a powerful counter to bacteria, they are ineffective against viruses. Often, bacterial and viral infections are confused due to their similar symptoms and lack of rapid diagnostics. With many clinicians relying primarily on symptoms for diagnosis, overuse and misuse of modern antibiotics are rife, contributing to the growing pool of antibiotic resistance.

Temporal modulation of the NF-κB RelA network in response to different types of DNA damage.

Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-κB transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA.

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics.

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion.

Strong anion exchange-mediated phosphoproteomics reveals extensive human non-canonical phosphorylation.

Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site-specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other "non-canonical" amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non-canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature.

Scanning Quadrupole Data-Independent Acquisition, Part B: Application to the Analysis of the Calcineurin-Interacting Proteins during Treatment of Aspergillus fumigatus with Azole and Echinocandin Antifungal Drugs.

Calcineurin is a critical cell-signaling protein that orchestrates growth, stress response, virulence, and antifungal drug resistance in several fungal pathogens. Blocking calcineurin signaling increases the efficacy of several currently available antifungals and suppresses drug resistance. We demonstrate the application of a novel scanning quadrupole DIA method for the analysis of changes in the proteins coimmunoprecipitated with calcineurin during therapeutic antifungal drug treatments of the deadly human fungal pathogen Aspergillus fumigatus.

Scanning Quadrupole Data-Independent Acquisition, Part A: Qualitative and Quantitative Characterization.

A novel data-independent acquisition (DIA) method incorporating a scanning quadrupole in front of a collision cell and orthogonal acceleration time-of-flight mass analyzer is described. The method has been characterized for the qualitative and quantitative label-free proteomic analysis of complex biological samples. The principle of the scanning quadrupole DIA method is discussed, and analytical instrument characteristics, such as the quadrupole transmission width, scan/integration time, and chromatographic separation, have been optimized in relation to sample complexity for a number of different model proteomes of varying complexity and dynamic range including human plasma, cell lines, and bacteria.

Evaluation of Parameters for Confident Phosphorylation Site Localization Using an Orbitrap Fusion Tribrid Mass Spectrometer.

Confident identification of sites of protein phosphorylation by mass spectrometry (MS) is essential to advance understanding of phosphorylation-mediated signaling events. However, the development of novel instrumentation requires that methods for MS data acquisition and its interrogation be evaluated and optimized for high-throughput phosphoproteomics. Here we compare and contrast eight MS acquisition methods on the novel tribrid Orbitrap Fusion MS platform using both a synthetic phosphopeptide library and a complex phosphopeptide-enriched cell lysate.

The mzIdentML Data Standard Version 1.2, Supporting Advances in Proteome Informatics.

The first stable version of the Proteomics Standards Initiative mzIdentML open data standard (version 1.1) was published in 2012-capturing the outputs of peptide and protein identification software. In the intervening years, the standard has become well-supported in both commercial and open software, as well as a submission and download format for public repositories.

The mzqLibrary--An open source Java library supporting the HUPO-PSI quantitative proteomics standard.

The mzQuantML standard has been developed by the Proteomics Standards Initiative for capturing, archiving and exchanging quantitative proteomic data, derived from mass spectrometry. It is a rich XML-based format, capable of representing data about two-dimensional features from LC-MS data, and peptides, proteins or groups of proteins that have been quantified from multiple samples. In this article we report the development of an open source Java-based library of routines for mzQuantML, called the mzqLibrary, and associated software for visualising data called the mzqViewer.

ProteoAnnotator--open source proteogenomics annotation software supporting PSI standards.

The recent massive increase in capability for sequencing genomes is producing enormous advances in our understanding of biological systems. However, there is a bottleneck in genome annotation--determining the structure of all transcribed genes. Experimental data from MS studies can play a major role in confirming and correcting gene structure--proteogenomics.

Numerical compression schemes for proteomics mass spectrometry data.

The open XML format mzML, used for representation of MS data, is pivotal for the development of platform-independent MS analysis software. Although conversion from vendor formats to mzML must take place on a platform on which the vendor libraries are available (i.e.

The utility of porous graphitic carbon as a stationary phase in proteomics workflows: two-dimensional chromatography of complex peptide samples.

We present the first investigation into the utility of porous graphitic carbon (PGC) as a stationary phase in proteomic workflows involving complex samples. PGC offers chemical and physical robustness and is capable of withstanding extremes of pH and higher temperatures than traditional stationary phases, without the likelihood of catastrophic failure. In addition, unlike separations driven by ion exchange mechanisms, there is no requirement for high levels of non-volatile salts such as potassium chloride in the elution buffers, which must be removed prior to LC-MS analysis.

Web-based design and evaluation of T-cell vaccine candidates.

Unlabelled: We present a suite of on-line tools to design candidate vaccine proteins, and to assess antigen potential, using coverage of k-mers (as proxies for potential T-cell epitopes) as a metric. The vaccine design tool uses the recently published 'mosaic' method to generate protein sequences optimized for coverage of high-frequency k-mers; the coverage-assessment tools facilitate coverage comparisons for any potential antigens. To demonstrate these tools, we designed mosaic protein sets for B-clade HIV-1 Gag, Pol and Nef, and compared them to antigens used in a recent human vaccine trial.

Polyvalent vaccines for optimal coverage of potential T-cell epitopes in global HIV-1 variants.

HIV-1/AIDS vaccines must address the extreme diversity of HIV-1. We have designed new polyvalent vaccine antigens comprised of sets of 'mosaic' proteins, assembled from fragments of natural sequences via a computational optimization method. Mosaic proteins resemble natural proteins, and a mosaic set maximizes the coverage of potential T-cell epitopes (peptides of nine amino acids) for a viral population.

Accurate on-line support vector regression.

Batch implementations of support vector regression (SVR) are inefficient when used in an on-line setting because they must be retrained from scratch every time the training set is modified. Following an incremental support vector classification algorithm introduced by Cauwenberghs and Poggio (2001), we have developed an accurate on-line support vector regression (AOSVR) that efficiently updates a trained SVR function whenever a sample is added to or removed from the training set. The updated SVR function is identical to that produced by a batch algorithm.