Getting Started with CHOP-Penn Proteomics Core

With the starting assumption that you have some reagent for affinity capture (i.e., immunoaffinity or other means) of the protein of interest.

1. With western blot or other means of detection find gentle lysis conditions that put most or all of the bait protein in solution. This can be done by probing gels lanes containing starting cells material, lysate, and lysis pellet while normalizing for volumes applied to the gel. Most or all of the target protein should be in the lysate. You will not be able to immunoprecipitate from the pellet.
2. When doing the affinity enrichment, see whether you've been able to capture all or most of the target protein. If a lot is left in the flow-through fraction then that may actually be what you're interested in (e.g., a post-translational modification or interacting protein may be occluding the affinity epitope on your target protein).
3. When 1 and 2 are done to your satisfaction, scale up to where the target protein is a clear band in a Coomassie stained gel or at least near the top of the list of proteins identified by mass spectrometry. Perform replicates and consider controls.

This varies by the cell or tissue type and the requirement of the proteomic questions being asked. For whole proteome analysis alone, one microgram to tens or hundreds of micrograms are a good place to start. Just how much depends on the analytical plans established during consultation. For OMIC-scale ptm analysis (i.e., phosphoproteome, ubiquitylome, lysine acetylome, glycoproteome) milligram quantities of protein are required. Other kinds of proteomic experiments are highly sample dependent. Targeted quantification often depends on the abundance (absolute and relative to other proteins) of the protein(s) of interest.

There are a variety of ways we communicate results to investigators:

• Tab-delimited text files containing many details of relating to protein and peptide identification and quantification
• Proteome Software Scaffold files
• Complete statistical and bioinformatic results with illustrative figures and detailed tables of results (advanced analysis may incur additional costs)

If is identified from the assembly of many unique peptide spectral matches of good intensity then almost definitely the answer is yes.

If it is identified by a poor quality MSMS spectral match to a low abundance ion the answer is maybe" Our false discovery threshold is typically set at 1 percent so that approximately 1 percent of the peptide and protein identification are incorrect. We just don’t know which ones those are.

Yes, please. After establishing a well-conceived experimental design, care and consistency of sample prep has a huge impact on the success of the analysis by mass spectrometry. Many upstream sampling practices create difficulties for the downstream proteomic analysis. We take great care to keep our “personal clouds” at bay to reduce keratin contamination and encourage our clients to do the same. It is best to consult with core staff before doing your experiments.

Every project the CHOP Proteomics Core provides service for requires the customized combination of wide-ranging itemized procedures. The extent of such combinations varies greatly depending on the nature of the samples and analytical goals of the project. For new collaborations and projects it is best to contact the core director for guidance on experimental design and sample procurement/delivery.

At this time and for other recurring sample submissions the core staff can provide a detailed estimate of the scope of work required along with associated costs and approximate time to completion. Because there are unique challenges associated with each sample type or project costs may vary depending on what additional procedures are required for successful completion of the analysis by mass spectrometry. Most of these additional considerations can be discussed with the investigator during initial consultation and at the time of sample submission.

We also serve University of Pennsylvania clients, providing the same CHOP-subsidized pricing for services. Outside academic and commercial clients receive 10 percent and 30 percent surcharges, respectively, while both are also charged for the quality control runs associated with each sample set.

This is highly variable depending on the scope of work involved, number of samples, instrument availability and downtime. Projects are done on a first come, first served basis ordered by the arrival of samples in the lab and when submission paperwork is completed. We can often fit short runs (i.e., one or two) in amongst longer multi-sample runs. Time estimates are provided at time of sample submission and updated as work progresses.

We have established and faithfully apply quality control procedures for almost every aspect of numerous established workflows.

We cannot guarantee your samples will work; however, if guidelines established during consultation are followed and established benchmarks met they have a very good chance. What we do guarantee is that the procedures we follow and instrument performance are all accomplished at a high, quality-controlled level.

That depends on the question you're asking. For rigorous statistical comparison of two or more cell conditions, three replicates is a bare minimum for each condition and five is becoming the new three. For clinical samples, the number is much higher and depends on the inherent variability of patients and sampling procedures.

First in, first out with the occasional exception for one or two samples that we can fit in between larger sample runs.

This needs to be discussed with core staff before the experiment is done and samples submitted. We have broad experience with a wide range of sample conditions and can often give sage advice to the investigator. We can devise tests for situations we haven't encountered before.

Publications containing data generated by the CHOP Proteomics Core should include acknowledgements to the CHOP Proteomics Core Facility and/or include members key to performing this work as co-authors. The latter can be justified by the creative problem solving that attends almost all but the most routine sorts of analyses we perform. Such acknowledgement and authorship adds an important component to our value proposition to help secure funding for our clients and for our core facility, and maintain our technical abilities near the leading edge of proteomics research.