Proteomics Mass spectometry technology center

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.  

Analysis options

click image to download .ppt

• Proteome analysis of cells or tissues

  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

• Immuno purifications/Affinity purifications/Purified Protein /Gel Slice

  This group of sample types contains all samples originating from pull down experiments, purified proteins and single gel slices. Compared to cells, tissues or body fluids these samples are less complex and can be analyzed with shorter runs (1 hour instead of 4 hour LC gradient). Depending on the origin of the samples and your ex-perimental design either in-solution digestion (Protocol 3) or in-gel digestion (Protocol 4) can be performed. Depending if only identification or also label-free quantification is needed, analysis is done on AmaZon Speed ETD MS (Analysis B) or maXis 4G+ MS (Analysis A). For the first data analysis is performed using Mascot as a database search engine and for the latter data analysis is performed using MaxQuant (database searching and Label Free Quantitation).

  Tips for successful pull down experiments

  • Many commercial kits for pull down experiments contain buffers with detergents, NP-40 or other pol-ymers. These substances disturb the LC-MS analysis. Therefore, the use of a detergent-removal col-umn is essential. Please, check the specifications of the detergent-removal column if the constituents of your buffers are effectively removed.
  • For optimal analysis of the proteins in your pull down, we recommend to choose an experimental set-up in which the antibody used for the pull down remains on the beads and does not elute into the sample. High amounts of antibody can suppress the signals of the proteins that are pulled down. Ways to limit the presence of antibody in the sample are:
    • Covalently binding the antibody to the beads
    • Using gentle elution buffers to leave antibodies intact and bound on beads

• Body fluids

  Most body fluids are very complex in proteome composition. Compared to samples originating from tissue or cell culture, body fluids (e.g. serum, plasma, CSF) have the drawback of containing large amounts of highly abundant proteins interfering with the analysis (e.g. albumin, transferrin). To remove these abundant proteins, a MARS-14 depletion column is used to deplete the top 14 most abundant proteins from the sample (Albumin, IgG, Transferrin, Haptoglobin, Alpha1-antitrypsin, Fibrinogen, Alpha2-Macroglobulin, Alpha1-Acid Glycoprotein, IgM, Apolipoprotein AI, Apolipoprotein AII, Complement C3, Transthyretin). This is followed by in-solution di-gestion (Protocol 5). Due to the complexity of the samples a 4 hour LC gradient is used in combination with MS analysis on the MaXis 4G+ MS (Analysis C) is used for either label-free quantification or identification. Identifi-cation and Label Free Quantitation is performed using MaxQuant.

Sample submission

Proteomics services

• Submission form

  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

• Shipment of samples

  Shipment of the samples to our laboratory can be done only after registration of your sample set (by sending us a copy of the sample submission form by e-mail).
  
  Internal customers
  Please make an appointment by email or phone (+31 (0)24 36 16933) to arrange sample transfer.
  
  External customers
  Send your samples on dry ice to the following address:
  
  Radboudumc
  Department of Laboratory Medicine
  815 -TML - Radboud Proteomics Center
  PO Box 9101
  6500 HB Nijmegen
  The Netherlands
  
  Please notify us when you ship samples to us, you will receive an email after arrival of your samples.

• Turnaround time

  Once your samples have arrived, it takes approximately 2 weeks to perform LC-MS and data analysis. Analyses on the UHR-QTOF MS (analyses A and C), may have a longer turnaround time. This instrument is heavily used. We will keep you updated on the progress in case there is a delay. If you want to make sure that your results are delivered as quickly as possible, it helps to notify us timely (at least 2 weeks before sample arrival) that you will be sending in samples. However, then it is essential that your samples are delivered at the arranged time!
  
  Additionally, if identification of proteins is the main goal and semi-quantitative information is not that important, you can choose for analysis B. This is performed using our ion trap MS system which has a lower occupancy than the UHR-QTOF MS, and thus a shorter turnaround time.

• Required additional information

  Alternative database
  For optimal database searches it is important that the database used contains all proteins that could possibly be present in your sample. Therefore, if your samples originate from other organisms than listed, or if you have expressed modified forms of a protein (including a tag, or originating from another organism), you have to provide us with the correct protein database to perform data analysis.
  
  The protein database has to be in FASTA format, and should contain all proteins from the organism and/or added non-standard protein sequences. Failing to deliver the FASTA protein database can delay the processing of the samples.
  
  Sample description
  For correct data analysis, it is important that we know how your experiment was designed. Please specify sample name, sample groups (f.e. control, patient) and fraction number (if your samples are fractionated).

• Costs for proteomics services

  By signing your sample submission form, you agree to the costs for the analysis of the samples. After finishing the proteomic analyses, we will send an invoice to you for the costs of the proteomics services.

• Storage

  Note that for publication purposes, raw data or additional processing data might be needed. Both raw LC-MS analysis files and data processing files are available on request. It is your own responsibility to store and archive these data for this purpose. We will store all data related to proteomics services for one (1) year after analysis. After this period, we will delete these data without prior notice.

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:

• In-solution digestion for cells/tissues

  You can download the protocol here.

• In-gel digestion for cell/tissues (+/- fractionation)

  You can download the protocol here.

• In-solution digestion for immunopurifications/affinity purifications/purified proteins

  You can download the protocol here.

• In-gel digestion for immunopurifications/affinity purifications/purified proteins

  You can download the protocol here.

• In-solution digestion protocol for human body fluids (plasma, serum, CSF)

  You can download the protocol here.

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.

• I want to perform LC-MS analyses myself using your infrastructure. Is that possible?

  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.

• I want to perform another proteomics approach such as targeted proteomics, intact protein analysis or analysis of post-translational modifications. Is that possible?

  Our proteomics services only offer-peptide based untargeted analyses. In our research projects, however, we do use these different approaches. Please contact us if you want to collaborate with us.

• I want to use the results of proteomics experiments for publication. Can you provide me with a description of the methods used?

  Yes, publication-ready descriptions of all service methods are available upon request. You can also send us a draft of your manuscript so we can verify that technical proteomics details are sound.
  
  All work provided by the Radboud Proteomics Center which is included in a publication should acknowledge the contribution of the RPC either in the acknowledgements section or as an author, depending our input. Proper acknowledgment of the RPC enables us to obtain financial and other support so that we may continue to provide essential services in the best ways possible. Please follow the ABRF Guidelines. Once the work is published, we appreciate a notification.  

• I want to use the results of proteomics experiments for publication. Which data do I need for publication?

  That depends on the type of journal you want to publish in. Some journals require data to be deposited in online repositories, and some do not ask for supplementary information. We have adapted the FAIR principles of data stewardship and make our own proteomics data sets available through PRIDE. We advice to store all raw LC-MS data and additional data processing files. We will store all data related to proteomics services for one year after analysis. After this period, we will delete these without prior notice. We do not deliver these files to you after finishing the analyses, but these files are available on request. It is your own responsibility to store and archive these data.

Experimental design

FAQ

• I want to see as many proteins as possible in my sample. How can I do that?

  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.

• I want a more extensive coverage of my body fluid proteome, can I further fractionate the sample after depletion?

  Yes, that is possible. It is possible to perform fractionation via SDS-PAGE after MARS-14 depletion. First perform the MARS-14 depletion from protocol 5, then you can load your depleted sample on SDS-PAGE and proceed with protocol 2. However, be aware that the costs of your experiment increase quickly when applying fractionation.

• You recommend MARS-14 depletion for body fluids. Are there alternatives?

  As the MARS-14 column is expensive, you can choose an alternative strategy, although most are not as effective as using the MARS-14 column. An alternative would be running the sample on gel and fractionate it (see protocol 2), with special care of excising the band around 65 kDa as this contains mainly albumin. This band will be analyzed but the more precise this band is excised, the fewer albumin will interfere in other fractions.

• Why would I choose analysis B as an option, with analysis A I also get quantitative results?

  In analysis B, the ion trap MS (amaZon Speed ETD) is used instead of the UHR-QTOF MS (maXis 4G+). This is a lower resolution instrument, and proteomics analyses using this instrument result in less accurate quantitation data and in complex samples generally a slightly lower number of identified proteins than analysis on a high resolution instrument. However, the occupation of the ion trap MS is lower than that of the UHR-QTOF MS. Therefore, analyses can be performed more quickly, and turnaround times are shorter.
  
  If the speed of the analysis is important and you want to make sure that your results are delivered as quickly as possible, it helps to notify us timely (at least 2 weeks before sample arrival) that you will be sending in samples. However, do ensure that your samples are delivered at the arranged time!

• How many technical and/or biological replicates do I need?

  The number of replicates depends mainly on the biological variation between your samples and sample groups. Unfortunately, we cannot predict how many replicates will be needed.
  
  The technical variation of the LC-MS analysis itself is very small compared to the biological variation. Therefore, we recommend to have multiple biological replicates.

• How long does it take until I get my results?

  Once your samples have arrived, it takes approximately 2 weeks to perform LC-MS and data analysis. Depending on the availability of the LC-MS instruments the turnaround time can be somewhat shorter or longer. We will keep you updated on the progress in case there is a delay. If you want to make sure that your results are delivered as quickly as possible, it helps to notify us timely (at least 2 weeks before sample arrival) that you will be sending in samples. However, do ensure that your samples are delivered at the arranged time!

Sample preparation

FAQ

• I don’t have all the required consumables and reagents for sample preparation. Can I get these from you?

  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.

• I don’t see a separate protocol for analysis of urine. Can I use the same protocol as for plasma/CSF?

  We have not tested the protocol for urine and, as of yet we have limited experience with analyzing the urinary proteome. From these studies we have seen that some urine constituents can influence the chromatographic separation. Therefore, we recommend to perform a protein precipitation prior to digestion. We do not have an optimized protocol for that, please check literature for this. After resuspending the protein pellet (make sure, the pH of the sample is okay!) you can proceed with either in-solution digestion (protocol 1) or in-gel digestion (protocol 2).

• I have done an immunoprecipitation and I suspect to have less than 10 μg proteine. I don’t want to spend any of the sample on a protein determination. Can I digest all and have this analyzed?

  We know that 10 μg is quite a lot, especially for IP or AP experiments. For these types of samples, digest the complete sample and send this in. Please, notify us that the amount of total protein might be low (on your sample submission form).

• What types of detergents can be used in combination with LC-MS analysis?

  Most detergents give rise to suboptimal chromatographic separation. However, these compounds might be needed for an optimal sample preparation. That is exactly the reason that we have implemented the use of a detergent removal column in our protocols. You can check the column specifications to see which detergents can be used safely and at which concentration.

• Do you have a protocol for pull down experiments?

  No, there are many different options to perform pull down experiments that work well. For optimal analysis of the proteins in your pull down, we recommend to choose an experimental setup in which the antibody used for the pull down remains on the beads and does not elute into the sample. High amounts of antibody can suppress the signals of the proteins that are pulled down. Ways to limit the presence of antibody in the sample are:

  • Covalently binding the antibody to the beads
  • Using gentle elution buffers to leave antibodies intact and bound on beads

• What buffer constituents do I need to avoid in pull down experiments?

  The use of polymer substances such as NP-40 in pull down experiments has long been a big problem in combination with LC-MS analysis. The polymers ionize very well and disturb the chromatographic separation and also ionization of peptides. We recommend to avoid using NP-40 in your experiments.
  
  If that is not possible, the use of Pierce detergent removal columns (protocol 3) also helps removing trace amounts of polymers, or another option might be to use SDS-PAGE as sample clean-up (protocol 4).

• Can you do the sample preparation for me? I’m willing to pay for this.

  Sample preparation takes considerable time, we may not have the capacity to perform your sample preparation for you. To keep prices for LC-MS analyses as low as possible we have designed our service workflow so that you can do this yourself.
  
  If it is really impossible for you to perform the sample preparation, please contact dr. Jolein Gloerich to discuss options.

Data analysis

FAQ

• I want to obtain information on post-translational modifications (PTMs). Can you do this?

  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.

• What type of data do I get from you?

  As output of the analyses, we will send you a short report describing the methods used and a short summary of the results. Additionally you will receive an excel file containing the detailed results.
  For Mascot analyses, the excel file contains at least 3 tabs:

  • Parameters - contains all search parameters used
  • Proteins - contains all identified proteins
  • Peptides - contains all identified peptides

  For MaxQuant analyses, the excel file contains at least 3 tabs:

  • MQ _settings - contains MaxQuant output parameters.txt with all MaxQuant settings
  • MQ_RAW - contains MaxQuant output proteingroups.txt
  • MQ_output - is a condensed for of MQ_RAW, only listing the first protein of a group, identification and quantitation values

  For more information on the output of MaxQuant, check the MaxQuant website or publications on MaxQuant.

• In my excel file with Maxquant results, I cannot find which peptides were identified? Where do I find this information?

  Indeed, this information is not in the result file, however, feel free to request all raw and processing files. Raw output of the MaxQuant analyses is provided in text files in the "combined" folder. Information on peptide identifications can be found in evidence.txt and peptides.txt.
  
  For more information please check the MaxQuant website.

• In my MaxQuant output I see that a specific isoform of a protein is listed. Is this the only isoform that was identified?

  We report the first protein of every protein group. You will have to check the MQ_RAW tab or the MaxQuant "protein groups.txt" output file. There you will see all proteins that are listed for a group. If a group lists multiple proteins, this means that all these proteins might be present in the sample, but no protein specific peptides (unique to one protein and not present in the other proteins) could be detected.