Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ

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.

/pdf/insights-into-the-regulation-of-protein-abundance-from-1nb4o968ef.pdf

Insights into the regulation of protein abundance from proteomic and transcriptomic analyses

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

The MaxQuant computational platform for mass spectrometry-based shotgun proteomics.

On the Dependency of Cellular Protein Levels on mRNA Abundance.

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.

https://science.sciencemag.org/content/sci/312/5771/212.full.pdf

Mass spectrometry and protein analysis.

Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents

Testosterone analysis by LC-MS/MS is becoming the analytical method of choice over immunoassays due to its specificity and accuracy. However, neutral steroid hormones possess poor ionization efficiency in MS/MS, resulting in insufficient sensitivity for analyzing samples with trace concentrations of the hormones. The method presented here utilizes a derivatization step involving a novel, permanently charged, quaternary aminooxy (QAO) reagent or MS-tag that reacts to the ketone functionality of testosterone and significantly enhances its ESI-MS/MS sensitivity. This derivatization method enabled quantitation of total testosterone in human serum (200 μL) with a lower limit of quantitation (LLOQ) of 1 pg/mL (3.47 pmol/L), total testosterone in dried blood spots (8–10 μL) with a LLOQ of 40 pg/mL, and free testosterone in serum ultrafiltrate (400 μL) with a LLOQ of 0.5 pg/mL. The linearity of each of the high sensitivity applications was maintained over a broad dynamic range of 1–5000 pg/mL for the serum sample...

LC-ESI-MS/MS Analysis of Testosterone at Sub-Picogram Levels Using a Novel Derivatization Reagent

In some embodiments, this invention pertains to isotopically enriched N-substituted piperazine acetic acids. In some embodiments, this invention pertains to methods for the preparation of isotopically enriched N-substituted piperazine acetic acids.

Isotopically enriched N-substituted piperazine acetic acids and methods for the preparation thereof

This invention pertains to labeling reagents, labeled analytes, including mixtures thereof, and fragment ions derived therefrom. This invention also pertains to methods for the production of labeling reagents, including N-substituted piperazine acetic acid based labeling reagents, labeled analytes, including mixtures thereof, and fragment ions derived therefrom as well as methods of the analysis of the labeled analytes.

Labeling reagents, labeled analytes, including mixtures thereof, and fragment ions derived therefrom and methods for the analysis thereof

In various aspects, the present teachings provide labeling reagents and sets of labeling reagents containing one or more heavy atom isotopes for the relative quantitation, absolute quantitation, or both, of hydroxylated compounds including, but not limited to, hydroxylated ring containing compounds, steroids and sterols. In various aspects, the present teachings also provide methods for the analysis hydroxylated compounds including, but not limited to, hydroxylated ring containing compounds, steroids and sterols my MS/MS methods, in particular using mass differential tags.

Subhakar Dey

Papers

Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents

LC-ESI-MS/MS Analysis of Testosterone at Sub-Picogram Levels Using a Novel Derivatization Reagent

Isotopically enriched N-substituted piperazine acetic acids and methods for the preparation thereof

Labeling reagents, labeled analytes, including mixtures thereof, and fragment ions derived therefrom and methods for the analysis thereof

Tagging reagents and methods for hydroxylated compounds