Mass spectrometers for proteomics


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dr. Jolein Gloerich

+31 (0)24 361 63 97
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What is proteomics?

Proteomics is the large-scale study of proteins.

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What is proteomics?

Proteomics is the large-scale study of proteins. The proteome is a set of proteins present in an organism, tissue, cell type or body fluid. The proteome is highly dynamic and differs between cell types or tissues and also changes over time in response to a large variety of factors that influence protein expression, post-translational modification and protein trafficking.  

Proteomics services

Overview services

We offer standard services for protein identification and relative quantitation using shotgun proteomics (relative comparison between samples based on label free quantitation). Here, you can find practical information on sample preparation and sample submission.

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Overview services

We offer standard services for protein identification and relative quantitation using shotgun proteomics (relative comparison between samples based on label free quantitation). Here, you can find practical information on sample preparation and sample submission. Furthermore, background information on the LC-MS/MS analyses and data analyses that are performed on your samples is provided. In case you cannot find the information you are looking for, please check the FAQ section or you can contact dr. Jolein Gloerich via e-mail.

We have developed standard workflows for analysis of different sample types such as body fluids, purified/enriched protein fractions or cell/tissue homogenates. Our sample preparation protocols have been tested and optimized for use in combination with our LC-MS/MS instrumentation. Furthermore, LC-MS/MS methods and data processing have been optimized to maximize the number of identified/quantified proteins.

If you want to make use of our services, you can design your experiment yourself. In Figure 1, all available analysis options are listed. The decisions you have to make about your experiment are listed in blue. In the analysis options section you can find more information that can help you in choosing the best experimental design. Once you’ve chosen an approach, in red the correct protocol for sample preparation and the type of analysis is shown. You can prepare your samples yourself using our protocols.

Information on delivery of samples can be found in the sample submission section. Samples can be submitted only of a sample submission form is used. You can deliver your LC-MS ready samples to us. You can indicate on the sample submission form which type of analysis you want to perform. After receipt of the samples, we will perform LC-MS/MS measurement and subsequent data analysis for protein identification and/or quantitation.

Analysis options

We can offer different kinds of analysis like: proteome analysis of cells or tissues, body fluids, and immuno purifications/affinity purifications/purified protein /gel slice.

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Analysis options

click image to download .ppt



  • There are several options for proteomic analysis of tissues or cell samples. The choice mainly depends on the desired depth of the proteome coverage. An experiment setup with fractionation covers a larger part of the proteome than without fractionation, and thus provides a more in-depth proteome coverage.

    Analysis without fractionation
    For proteome analysis without fractionation, the simplest approach is an in-solution digestion (Protocol 1) of the sample. Alternatively, if an additional clean-up step is necessary or the sample is already in gel, you can choose to in-gel digest the sample (Protocol 2). Here, SDS-PAGE is not used as a separation technique but as a clean-up step. The complete sample is digested in one fraction, therefore we advice to only shortly run the sample on an SDS-PAGE to limit the volume of gel pieces for digestion.

    As the complexity of an unfractionated cell/tissue sample is very large, LC-MS analysis is performed using a 4 hour LC gradient combined with MS analysis on the MaXis 4G+ MS (Analysis C) for both in-gel and in-solution digested samples. Data analysis is performed using MaxQuant software (database searching and Label Free Quantitation). Database searches are performed using the protein database specified in the sample submission form.

    Analysis with fractionation
    For a more in-depth proteome profiling analysis, SDS-PAGE separation can be used to fractionate the sample by cutting each gel lane into multiple slices. Each slice is then separately in-gel digested (Protocol 2). LC-MS analy-sis is performed using a 1 hour LC gradient combined with MS analysis on either the MaXis 4G+ MS for identifi-cation and quantitation (Analysis A) or, if only protein identification is needed, on the AmaZon Speed ETD MS (Analysis B). Data analysis is performed with MaxQuant (identification and Label Free Quantitation) or Mascot (identification), for analysis A and B respectively.

    NOTE: Be aware that the number of analyses is the actual amount of vials handed in. Therefore, proteome profiling of 1 sample in 10 fractions


Sample submission

Information on shipment, turnaround time, storage and costs.

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Sample submission

Proteomics services


  • After you have made an experimental design for your proteomics experiment (please check Overview and Analysis options in the Proteomics services section for more information) and you have prepared your samples according to one of our protocols (see Protocols in the Proteomics Services section for more information), you can submit your samples for LC-MS analysis. To submit your samples, pick the right sample submission form (internal: Radboud University Medical Center, Radboud University, External: all other customers) and complete the form. Send one copy of the form by e-mail to register your sample set and include one printed and signed copy with the samples.

    Submission form for internal customers
    Submission form for external customers


Protocols

After you have made an experimental design for your proteomics experiment, you can start your sample preparation using our protocols.

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Protocols

Sample preparation protocols

After you have made an experimental design for your proteomics experiment (please check Overview and Analysis options in the Proteomics services section for information) you can start your sample preparation using our protocols. Each protocol lists required reagents and consumables. For internal customers (Radboud University Medical Center, Radboud University) we can provide certain reagents and/or consumables (Digestion enzymes Trypsin and LysC, C18 OMIX tips, Detergent removal column). If you want to make use of this option please fill out a sample submission form and contact us via e-mail to pick up your order, of course you can also order them yourself.

Below you find the 5 different sample preparation protocols to prepare your samples:


  • You can download the protocol here.

Proteomics experts

Frequently asked questions


General

All information on this website is meant to help you figure out how to perform a proteomics experiment. Our proteomics services are designed for small to medium scale proteomics experiments.

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General

FAQ

All information on this website is meant to help you figure out how to perform a proteomics experiment. Our proteomics services are designed for small to medium scale proteomics experiments. More specialized or large scale proteomics studies are executed as dedicated scientific projects. If you are unsure how to design your experiment after reading the information on our website, or need additional help we offer consultancy based on an hourly fee.

In the figure below, standard data analysis options offered by RTC Bioinformatics for differential proteome profiling data are shown. For additional questions how to perform bioinformatic or statistic analyses, we refer to the Radboudumc Technology Center of Bioinformatics or the Technology Center of Biostatistics.


  • That is possible, but only for larger projects (>6 months) in collaboration with our group to ensure sufficient training. Please contact us to discuss options and budget. Preferably, we are involved in grant proposals in an early stage, so that we can think with you how to set up the project and estimate costs.



Experimental design

FAQ


  • If your sample is complex, it helps to fractionate the sample in multiple fractions that can be analyzed separately. However, be aware that the size and costs of your experiment rise quickly when applying fractionation: Fractionating 1 sample into 10 fractions will result in 10 LC-MS analyses.

    Another option that helps to increase proteome coverage in body fluid samples (tested for plasma and CSF) is depletion of the most abundant proteins using the Agilent MARS-14 column.



Sample preparation

FAQ


  • We prefer that you buy all required items yourself, but we also understand that some package sizes are quite big, especially if you only have a small number of samples. Therefore, for internal customers (Radboud University Medical Center, Radboud University) we can provide certain reagents and/or consumables (Digestion enzymes Trypsin and LysC, C18 OMIX tips, Detergent removal column). If you want to make use of this option please fill out a sample submission form and contact dr. Jolein Gloerich via e-mail to make an appointment to pick up your order at the Radboud Proteomics Center.



Data analysis

FAQ


  • You have to specify which modification you want to study. In our database search we will have to include this modification, to identify peptides with this modification. This works for modifications that remain stable when using collision induced dissociation (f.e. methylation, acetylation). Keep in mind that we use a general LC-MS method and that might not be the best way to study specific modifications.

    If you want to perform detailed analysis of a specific PTM, often sample preparation protocols and sometimes LC-MS methods have to be adapted for this purpose. This is not offered as a service. Please contact us to discuss options for collaboration.

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