Dataset Information

The impact II, a very high resolution quadrupole time-of-flight instrument for deep shotgun proteomics

ABSTRACT: Hybrid quadrupole time-of-flight (QTOF) is one of the two major mass spectrometric technologies used in proteomics. Although based on simple fundamental principles, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated LC system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high MS/MS frequencies. The new reflectron and detector improve resolving power compared to the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4,800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2>0.99) and accurate fold change determination in spike-in experiments over three orders of magnitude in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originated from different tissues. Finally, after high pH reversed-phase fractionation we identified 9,515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in cerebellum â€“ the highest proteome coverage measured with a QTOF instrument so far.

INSTRUMENT(S): Bruker Daltonics instrument model

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast) Mus Musculus (mouse)

TISSUE(S): Embryonic Fibroblast, Lung, Cell Culture, Hela Cell

SUBMITTER: Mario Oroshi

LAB HEAD: Matthias Mann

PROVIDER: PXD001592 | Pride | 2015-05-26

REPOSITORIES: Pride

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Publications

The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics.

Beck Scarlet S Michalski Annette A Raether Oliver O Lubeck Markus M Kaspar Stephanie S Goedecke Niels N Baessmann Carsten C Hornburg Daniel D Meier Florian F Paron Igor I Kulak Nils A NA Cox Juergen J Mann Matthias M

Molecular & cellular proteomics : MCP 20150519 7

Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very ...[more]

PMID: 25991688

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Project description:we aimed to provide an objective roadmap for histone sample preparation wherein the most notable findings and most important metrics were highlighted. This roadmap included a Histone Coverage Tool that allows researchers to make a snapshot of public or in-house data in search of a protocol that suits their specific needs. However, despite the fact that the coverage provided by a sample preparation workflow is pivotal to answer specific research questions, it should be noted that other metrics such as enzyme specificity and workflow variability can be equally important. Moreover, when it comes to histone sample preparation there are multiple additional challenges and pitfalls compared to regular bottom-up proteomics workflows. First there is the abundance and unequal distribution of arginine and lysine cleavage sites, which makes it virtually impossible to cover the entire histone code with a single bottom-up workflow. The most widely spread workaround for the abundance of cleavage sites is derivatization of lysine residues used to block tryptic digest and create longer peptides. However, this also introduces chemical noise, which obscures biological PTMs, while lowering sensitivity due to diluted signal. The most obvious alternative is an arginine specific digest. However, on the one hand, derivatization of lysine residues not only increases peptide length but also increases retention on the LC-system, while decreasing charge state distribution of the precursor ions. The latter presumably reduces instrumental variation of the workflow, which is remarkable since this source of variation is generally assumed to be constant in the field of LC-MS/MS based proteomics. On the other hand, lysine derivatization also increases the singly charged precursor fraction, which is usually not fragmented in regular data dependent acquisitions, thus increasing the risk of losing annotations. It is clear that not one histone sample preparation workflow will fit all research questions. However, we hope that this manuscript will guide researchers in making informed decisions about the many pitfalls and trade-offs involved in optimizing or assessing histone sample preparation workflows.

Dataset Information

The impact II, a very high resolution quadrupole time-of-flight instrument for deep shotgun proteomics

Publications

The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics.

Similar Datasets

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