Core Facility — Proteomic Mass Spectrometry
Services
Acknowledgements
All work provided by the Core Facility - Proteomic Mass Spectrometry that is included in a publication record should include the following acknowledgement:
This work was supported by ZMG Core Facility Proteomic Mass Spectrometry - Martin-Luther-University Halle-Wittenberg.
It would be appreciated if you sent a copy of the accepted manuscript, published paper, or title of grant application to Matt Fuszard
In addition, if any facility personnel makes a substantial intellectual and/or experimental contribution to a publication recognition as any other co-author is expected.
This work was supported by ZMG Core Facility Proteomic Mass Spectrometry - Martin-Luther-University Halle-Wittenberg.
It would be appreciated if you sent a copy of the accepted manuscript, published paper, or title of grant application to Matt Fuszard
In addition, if any facility personnel makes a substantial intellectual and/or experimental contribution to a publication recognition as any other co-author is expected.
The CF offers World-class facilities for the analysis of peptides. A single purified protein in mixture or gel band can be trivially identified using our MALDI TOF/TOF searched with the Mascot algorithm. However our instruments employ profound sensitivity – the Waters G2S QTOF is equipped with StepWave (for more informatrion, please visit the link on the left site) technology which removes uncharged ‘masking’ ions, alongside ion mobility (for more informatrion, please visit the link on the left site) which separate identically eluting analytes – allowing for limits of sensitivity down to the attamole level. Instruments such as the Q-Exactive plus also enjoy phenomenal resolution and mass accuracy which is vital for reliable identification. Additionally we run ‘front-end’ nanoflow liquid chromatography solutions running either 10 cm (packed emitter) or 25 cm (nanoAcquity, pre-emitter) C18 columns for precise separation and additional sensitivity. This allows for us to maximise peptide coverage in simple mixtures, and, using a range of gradient lengths, reliably identify thousands of proteins in complex lysates. Additionally we have and use protocols for unusual and ‘difficult’ proteins (such as can be found in membranes, or with close-to-indigestible domains) using alternative proteases, C8 columns in middle-down proteomics workflows, various dissociation technologies and non-standard sample conditions to maximise our capability of characterising almost any protein with maximum efficiency. The final, but crucial, step in the analysis is the Search Engine. In this we have the best: alongside the well-known, and feature-packed 24-core Mascot platform (which will be free for registered users), we also run a 16-core Byonic sever, and an 8-core PEAKS DB (utilising so-called ‘hybrid’ algorithms) – both of which out-performed all other search engines in a recent iPRG study (for more informatrion, please visit the link on the left site). These engines will run in parallel to maximise confidence, and their respective results files blended together using Scaffold software (an integrative proteomics package and results viewer). Using the appropriate databases also, GO repositories can further be integrated to help compartmentalise confidently identified proteins with regard to cell location and function.
Intact Protein Analysis
In many cases, the analysis of intact proteins can offer significant advantages over peptide analytics. For example, if a protein is modified in a very hydrophobic region resistant to a C18 workflow, this mass shift can be trivially measured on the intact protein itself. In fact distributions of modifications (such as surface acetylation, or internal crosslinks) can be assessed and visualised, and alterations to the modification status of a protein can be monitored. N-terminal protein degradation, which is almost impossible to see accurately in a gel, let alone at the peptide level, becomes a routine analysis. Other advantages include being able to identify subtle changes to a protein, such as a point mutation, which had not been accounted for in a database entry as well as non-subtle shifts such as complex glycosylation which is also impossible with a peptide-based workflow. A variation of Intact Mass analytics, called Native Protein analysis due to the non-denaturing sample handling and injection process, is capable of assessing, directly, protein-protein interactions and subunit stoichiometry. An entire field of mass spectrometry called Top-Down Proteomics is dedicated to applying fragmentation energies to native protein clusters in order to provide solid MSMS evidence of protein interaction. The core facility has a range of both high and low resolution instruments which can provide high accuracy information about your intact protein. In addition we have the deconvolution software ‘Intact Mass’ (from Protein Metrics), which is vendor neutral, works with all levels of resolution, and – amazingly – independently processes any amount of elution peaks, completely automating a once time-consuming task.
PTM Characterisation & Localisation
Protein post-translational modification (PTM) is a crucial mechanism of cell regulation. To modify the function or interactions of proteins they can be modified by adding or removing chemical groups from certain amino acids. Additionally, PTMs of one protein can interact with each other, which leads to specific functions of the protein known as PTM cross-talk. We offer theoretical and practical state of the art characterization of different PTMs, such as phosphorylation, acetylation and glycosylation.
Quantitation
The Core Facility is equipped with multiple instruments which are capable of Shotgun-based High-Resolution, labelled (DDA - iTRAQ, TMT), SILAC, and label-free (DIA – HDMSE, MSn) and targeted (SRM, MRM & PRM) quantitative methods. These workflows are supported by a wealth of bioinformatics tools, centred around Scaffold Q+S, PLGS, ProteomeDiscoverer, Skyline and MaxQuant. Statistical inference and data analysis are handled through Perseus and multiple plug-in tools for OpenMS, Skyline, and PD written in R (such as MSstats). Indeed, workflows are envisaged that combine discovery DDA/DIA quantitative methods with very high sensitivity validation runs (MRMs), in order to maximise certainty, especially with regard to low fold-change observations.
Protein-Protein Interactions
Protein interaction maps are critical to fully understand protein function and structure. Individual proteins often exist in multi-mers and complexes, or react transiently with substrates within signalling pathways. Understanding these interactions, and their malfunctions, either temporally or spatially are a vital tool in understanding disease. The Core Facility is fully equipped to undertake protein interaction experiments for example through Co-Immunoprecipitation experiments, or through working closely with – Prof. Andres Sinz’s lab in the use of crosslinks. Bioinformatics is supplied through the use of Byonic, MassAI and (developed within AG Sinz) StavroX.
Small molecule/Peptide analytics
Our Bruker ultrafleXtreme MALDI-TOF/TOF is the ideal instrument to identify and characterise small molecules purified peptides, and low-complexity peptide mixtures.