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Sample Characterization – Qualitative Proteomics
An increasingly common request for proteomics core facilities is determining qualitative and quantitative differences among clinical samples such as plasma, CSF, or urine. PME6 was designed to test the ability of member laboratories to use mass spectrometry and bioinformatics tools for characterization (qualitative) of plasma proteins. This study is useful for laboratories to benchmark themselves against other groups, and detect areas for further improvement of their methodology. In an attempt to represent a realistic experiment scenario that might be requested to a proteomics core facility, we are providing a simple but challenging middle complexity protein sample, namely ASS17v3 sample. This sample is simple in the sense that the number of proteins is between 100 and 300 proteins depending on how you perform your protein inference; nevertheless, participating laboratories will face challenges regarding the sample dynamic range, the presence of protein isoforms and PTMs (mainly glycosilation and phosphorylation).
Study Scenario:
Dr. House received a limited grant to perform a biomarker discovery study on a subset of plasma proteins obtained by his immunoprecipitation method. Before hiring a proteomics core facility for this study, he skeptically decided to evaluate your ability to: (1) detect low abundance proteins, (2) discriminate protein isoforms, (3) provide improved dynamic range without peptide/protein pre-fractionation, (4) being reproducible, and (5) reach good sensitivity without degenerating specificity. For this purpose, Dr. House prepared a ‘demo’ sample and sent to your lab for analysis. He demands (Dr. House is very nagging) you to perform the experimental analysis at least 3 times in order to prove that your analysis pipeline is reproducible. In other words, he requires an experiment triplicate with three distinct digestions followed by its MS analysis. Due to the budget limitation and the low amount of sample obtained in each immunoprecipitation procedure, Dr. House requests a single MS analysis for each replicate sample. The sample therefore should not be pre-fractionated before LC-MS/MS analysis. Each triplicate should be provided as a single instrument run. For most instruments, this means that one single MS file (thermo.raw, ABI.wiff) or folder (waters.raw folder, ABI.t2d folder) is generated for each replicate. Dr. House’s reputation is well known. We learned from our good friend Dr. Wilson that House spiked 4 proteins in the sample, at different concentrations and spanning 5 orders of magnitude. 1433_HUMAN CASB_BOVIN PYGM_RABIT ALDOA_RABIT
Sample:
* Each participant laboratory is receiving an eppendorf tube (low-binding tube) containing 37.5 µl of the ASS17v3 sample. * You should perform your own quantitative assay in order to determine the protein concentration in the sample. For your guidance, we anticipate that the concentration of sample is between 1 and 3 mg/ml. You should provide the results of your quantification assay (regression curve, error, absolute values of quantification) on the results template (not available yet).  * Proteins are suspended in 50mM Tris.HCl pH 7.4. * Samples have been stored at -20ºC.
Specific problems to focus on:
(1) the identification of unknown proteins and discrimination between closely related protein isoforms; (2) reaching maximal sensitivity and specificity in terms of protein identification; (3) improving low abundance protein detection and dynamic range.
Methodology
- Sample complexity has been chosen to allow for the analysis of the mixture on single LC-MS runs. In principle, there is no need for pre-fractionation. A long enough gradient (90-120 min) gradient is suggested, but this of course will strongly depend on the MS instrument available for analysis. - The total amount of peptides injected on column will strongly depend on your LC-MS system. Using a nanoLC configured with pre-column + column (0.075 x 10 mm) coupled to a Velos, we have observed that injections of 200 nanograms is close to saturate the column. Again, you should determine the amount of sample injected on column for your system. Nevertheless, please keep track of this data (amount of digested protein injected on column) since it also required for reporting. - The amount of spiked proteins per microgram of total protein ranges from easy (high femtomoles) to quite hard (high attomoles) to be detected.
Centralized data analysis and MIAPE
Together with Salvador Martínez de Bartolomé and Alex Campos, who will be coordinating this, we will send you soon a separate document on how to analyze data and report protocols and results. The general idea is performing an “In-lab” analysis, with the lab’s own tools, and a second “centralized” analysis, by sharing the data in standard formats and using different software tools available for comparison. We will compile descriptions of the experimental methods that were used (in the MIAPE form) and highlight the more successful methods in terms of proteome coverage, improved dynamic range and high sensitivity/specificity. This information will be presented at the 2010 ProteoRed meeting.
Optional sample analysis optimization
We also welcome efforts on optimizing any technical aspect of proteomics analysis. If your laboratory is willing to use this sample as a standard to optimize proteomics techniques, we will have time slots for such presentations at the 2010 ProteoRed meeting. We’d greatly appreciate methodological optimization in different fields of proteomics analysis: sample preparation; glycoproteomics and phosphoproteomics analysis; mass spectrometry methods for increased proteome coverage, depth of analysis and dynamic range; improved chromatography techniques; bioinformatics strategies; and so on.   We foresee the use of this sample as a standard not only for optimizing methodologies, but also for performing quality control in proteomics laboratories. The PCB has been working on this sample during the past year. If you want to accept this challenge and want more information about the sample, please contact Alex Campos (acampos@pcb.ub.es; +34 934034653).
Dates of interest:
	Date of sample dispach: 19th-23rd July, 2010.
	Deadline for submission of results: 22nd October, 2010
	Proteored meeting scheduled on 18th November in Madrid where the results will be presented/discussed. Information about the meeting will be posted here
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