Author
Philip L. Ross
Other affiliations: Applied Biosystems
Bio: Philip L. Ross is an academic researcher from AstraZeneca. The author has contributed to research in topics: Avibactam & Matrix (chemical analysis). The author has an hindex of 14, co-authored 21 publications receiving 6384 citations. Previous affiliations of Philip L. Ross include Applied Biosystems.
Papers
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TL;DR: It is found that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression, and the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards.
4,411 citations
TL;DR: A model multiplex PCR system is demonstrated to have the ability to perform 12-fold multiplex SNP analysis and mass spectra of interleaved genotyping products can be generated with no ambiguity in allele assignment.
Abstract: A primer extension assay is used to perform highly multiplexed genotyping of single nucleotide polymorphisms (SNPs) present in genomic DNA amplified by a multiplex PCR. The assay uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry to accurately measure the masses of short oligonucleotide primers extended by a single dideoxynucleotide. The multiplexed genotyping assays rely on the natural molecular weight differences of DNA bases. By careful analysis of primer composition complementary to the target, or by judicious addition of one or more noncomplementary 5´ bases to the genotyping primers, mass spectra of interleaved genotyping products can be generated with no ambiguity in allele assignment. Using a model multiplex PCR system, we demonstrate the ability to perform 12-fold multiplex SNP analysis.
486 citations
TL;DR: It is shown that avibactam is a covalent, slowly reversible inhibitor, which is a unique mechanism of inhibition among β-lactamase inhibitors.
Abstract: Avibactam is a β-lactamase inhibitor that is in clinical development, combined with β-lactam partners, for the treatment of bacterial infections comprising Gram-negative organisms. Avibactam is a structural class of inhibitor that does not contain a β-lactam core but maintains the capacity to covalently acylate its β-lactamase targets. Using the TEM-1 enzyme, we characterized avibactam inhibition by measuring the on-rate for acylation and the off-rate for deacylation. The deacylation off-rate was 0.045 min−1, which allowed investigation of the deacylation route from TEM-1. Using NMR and MS, we showed that deacylation proceeds through regeneration of intact avibactam and not hydrolysis. Other than TEM-1, four additional clinically relevant β-lactamases were shown to release intact avibactam after being acylated. We showed that avibactam is a covalent, slowly reversible inhibitor, which is a unique mechanism of inhibition among β-lactamase inhibitors.
415 citations
TL;DR: An 8‐plex version of an isobaric reagent for the quantitation of proteins using shotgun methods is presented and it is found that there are differences in considering peptide‐based observations versus protein-based observations from quantitative shotgun proteomics studies.
Abstract: An 8-plex version of an isobaric reagent for the quantitation of proteins using shotgun methods is presented. The 8-plex version of the reagent relies on amine-labeling chemistry of peptides similar to 4-plex reagents. MS/MS reporter ions at 113, 114, 115, 116, 117, 118, 119, and 121 m/z are used to quantify protein expression. This technology which was first applied to a test mixture consisting of eight proteins and resulted in accurate quantitation, has the potential to increase throughput of analysis for quantitative shotgun proteomics experiments when compared to 2- and 4-plex methods. The technology was subsequently applied to a longitudinal study of cerebrospinal fluid (CSF) proteins from subjects undergoing intravenous Ig treatment for Alzheimer's disease. Results from this study identify a number of protein expression changes that occur in CSF after 3 and 6 months of treatment compared to a baseline and compared to a drug washout period. A visualization tool was developed for this dataset and is presented. The tool can aid in the identification of key peptides and measurements. One conclusion aided by the visualization tool is that there are differences in considering peptide-based observations versus protein-based observations from quantitative shotgun proteomics studies.
341 citations
TL;DR: Kinetic parameters of inhibition as well as acyl enzyme stability are reported against six clinically relevant enzymes, providing the foundation for future structural and mechanistic enzymology experiments.
293 citations
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TL;DR: A new intensity determination and normalization procedure called MaxLFQ is developed that is fully compatible with any peptide or protein separation prior to LC-MS analysis, which accurately detects the mixing ratio over the entire protein expression range, with greater precision for abundant proteins.
3,732 citations
TL;DR: Current understanding of the major factors regulating protein expression is summarized to demonstrate a substantial role for regulatory processes occurring after mRNA is made in controlling steady-state protein abundances.
Abstract: Recent advances in next-generation DNA sequencing and proteomics provide an unprecedented ability to survey mRNA and protein abundances. Such proteome-wide surveys are illuminating the extent to which different aspects of gene expression help to regulate cellular protein abundances. Current data demonstrate a substantial role for regulatory processes occurring after mRNA is made - that is, post-transcriptional, translational and protein degradation regulation - in controlling steady-state protein abundances. Intriguing observations are also emerging in relation to cells following perturbation, single-cell studies and the apparent evolutionary conservation of protein and mRNA abundances. Here, we summarize current understanding of the major factors regulating protein expression.
3,308 citations
TL;DR: An updated protocol covering the most important basic computational workflows for mass-spectrometry-based proteomics data analysis, including those designed for quantitative label-free proteomics, MS1-level labeling and isobaric labeling techniques is presented.
Abstract: MaxQuant is one of the most frequently used platforms for mass-spectrometry (MS)-based proteomics data analysis Since its first release in 2008, it has grown substantially in functionality and can be used in conjunction with more MS platforms Here we present an updated protocol covering the most important basic computational workflows, including those designed for quantitative label-free proteomics, MS1-level labeling and isobaric labeling techniques This protocol presents a complete description of the parameters used in MaxQuant, as well as of the configuration options of its integrated search engine, Andromeda This protocol update describes an adaptation of an existing protocol that substantially modifies the technique Important concepts of shotgun proteomics and their implementation in MaxQuant are briefly reviewed, including different quantification strategies and the control of false-discovery rates (FDRs), as well as the analysis of post-translational modifications (PTMs) The MaxQuant output tables, which contain information about quantification of proteins and PTMs, are explained in detail Furthermore, we provide a short version of the workflow that is applicable to data sets with simple and standard experimental designs The MaxQuant algorithms are efficiently parallelized on multiple processors and scale well from desktop computers to servers with many cores The software is written in C# and is freely available at http://wwwmaxquantorg
2,811 citations
TL;DR: It is concluded that transcript levels by themselves are not sufficient to predict protein levels in many scenarios and to thus explain genotype-phenotype relationships and that high-quality data quantifying different levels of gene expression are indispensable for the complete understanding of biological processes.
1,996 citations
TL;DR: Recent advances in mass spectrometry instrumentation are reviewed in the context of current and emerging research strategies in protein science.
Abstract: Mass spectrometry is a central analytical technique for protein research and for the study of biomolecules in general. Driven by the need to identify, characterize, and quantify proteins at ever increasing sensitivity and in ever more complex samples, a wide range of new mass spectrometry-based analytical platforms and experimental strategies have emerged. Here we review recent advances in mass spectrometry instrumentation in the context of current and emerging research strategies in protein science.
1,992 citations