Proteomics FAQs

Do you accept off-campus samples?

Yes, we certainly do. Please contact hremmer@umich.edu for more details.

What is your turnaround time?

For Proteomics and protein analysis projects: usually 5-15 business days depending on the type of service.

For Peptide Synthesis projects: usually 2-3 weeks.

For Pathway Analysis: 1-4 business days.

How do your prices compare to other Cores?

Our prices are listed on our website. We think that our prices are comparable to other Cores and competitive. Some of our prices may appear high, as we include sample preparation and reasonable fractionation for many of our proteomics options. Please inquire and/or schedule a consultation by contacting the Core.

Co-Authorship

If you think that we contributed intellectually to your project it is appropriate to list us as co-authors. If you want a more detailed explanation of authorship when using a core facility please have a look at the ABRF Authorship Guidelines. These are the standards of the Proteomics Community most core facilities follow when there are questions about authorship.

What instrument should I choose for protein ID?

We offer two price options for protein ID. Medium to strongly coomassie stained gel bands or proteins in solution with concentrations of at least 20 micromolar, can be analyzed on the Orbitrap Velos Pro for a lower price. All other samples containing significantly less or little protein should be analyzed on the Q-Exactive or Fusion Lumos Instruments.

Do you have protocols for sample preparation?

Yes, we usually discuss sample preparation tailored to the project during the consultation, in person, by phone or email. In general, you may find useful of protocols, buffers and kits on the ThermoFisher web site. For highly complex samples and clinical samples we prefer to perform sample preparation at the Core and highly recommend submission of frozen cells/tissue/blood plasma or other biological or clinical sample formats. Please contact the Core in regards to sample quantity and special protocols.

What is Scaffold?

Scaffold is a user-friendly software platform to visualize and validate MS/MS proteomics experiments.  This versatile software is available for download as a free viewer. Training is available at the Core. The free viewer allows you to identify proteins intuitively and confidently, compare samples to identify biological relevance, identify protein isoforms and localize protein posttranslational modification (PTMs), drill down into the MS/MS spectra and spectral counts, create comprehensive lists of target proteins, classify proteins according to Gene Ontology terms, quantitate proteins by spectral counting (label-free) and TMT.

How do I view and interpret data using the Scaffold software?

When you obtain results from the Core, you will typically obtain 3 data files, a project report with experimental conditions and the results summary (word document), a list of identified proteins with contaminants removed (in excel) and the Scaffold file. When we discuss your results, you will be trained to use the Scaffold viewer, so you can extract the most information from your data. The Proteome Software website also has tutorials on how to use Scaffold.

Do you offer any seminars e.g. to help with data interpretation?

Yes. In addition to individual software training and data interpretation, we will be happy to come to your research group meeting or department meeting to give a concise presentation on proteomics topics of your interest. We also organize regular seminars (2-3 per year) where we invite speakers to present on relevant and trending proteomics topics related to new services.

What is the difference between all the options in quantitative proteomics?

There are two ways to quantitate proteins by mass spectrometry, label-free and labeled.

Label-free quantitation uses the spectral count (number of MS/MS spectra assigned to an identified protein) for relative quantitation. This method is used in protein expression profiling and yields the “fold-change” of a given proteins between samples or categories of samples. Under labeled quantitation we offer metabolic labeling (stable isototope labeling by amino acids in cell culture, SILAC) and quantitation by Tandem Mass Tags (TMT) , both yield relative quantitation using peak area. The most accurate method is quantitation using  a heavy isotope-labeled internal standard, as done in Parallel reaction monitoring, PRM (or SRM, MRM).

For the need of replicates, please see that topic in our FAQ.

How many replicates do I need for reliable quantitation?

If you want to use label –free proteomics as stand-alone method of quantitation, 3 replicates (n=3) are necessary. Alternatively, you can use n=1 and validate the quantitation using an orthogonal method such as western blotting or parallel reaction monitoring (PRM) by mass spectrometry. In a TMT experiment, no replicates are necessary, n=1 is sufficient although for some experiments, duplication or triplication is recommended. No replicates are needed for PRM experiments, since quantitation happens based on an internal standard.

What is Pathway Analysis and what type data do I need to have?

Pathway Analysis (also known as functional enrichment analysis) is used to analyze data obtained from proteome expression profiling or other proteomics experiments.  Pathway Analysis relates the statistical analysis of the data set with biological function by identifying the biochemical pathway and how the differentially expressed proteins influence other proteins or pathways. This can lead to the identification of additional target proteins. The software we use is the iPathway guide by Advaita Bioinformatics. We usually upload Scaffold data, but can also work with any data set in excel format that contain a protein ID, a p-value and a fold-change.

Which components in my sample do I need to avoid for good mass spectrometry data?

In general: detergents, glycerol and urea (>2M). For mass analysis of intact proteins, we list acceptable and non-acceptable buffer components on our request form.