Proteins & Mass Spectrometry

MALDI TOF Mass spectrometry and tandem MS/MS

2D LC-MALDI Separation and Analysis Procedures

2D-LC separations:

After iTRAQ labeling is complete, or for LC-MALDI MudPit experiments, 2D-LC separation of the tryptic peptides is carried out as follow:

The samples are dried down and resuspended in SCX loading buffer (Buffer A below).

SCX Separations are performed on a passivated Waters 600E HPLC system, using a 4.6 X 250 mm PolySULFOETHYL Aspartamide column (PolyLC, Columbia, MD) at a flow rate of 1 ml/min. Buffer A contains 10 mM ammonium formate, pH 3.6, in 20% acetonitrile/80% water. Buffer B contains 666 mM ammonium formate, pH 3.6, in 20% acetonitrile/80% water.

The gradient is Buffer A at 100% (0- 22 minutes following sample injection), 0%→40% Buffer B (22-48 min), 40%→100% Buffer B (48-49 min) 100% Buffer B isocratic (49-56 min), then at 56 min switched back to 100% A to re-equilibrate for the next injection. The first 28 ml of eluant (containing all flow-through fractions) are combined into one fraction, then 14 additional 2-ml fractions are collected. All 15 of these SCX fractions are dried down completely to reduce volume and to remove the volatile ammonium formate salts, then resuspended in 9 µl of 2% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid and filtered prior to reverse phase C18 nanoflow-LC separation.

For 2nd dimension separation by reverse phase nanoflow LC, each SCX fraction is autoinjected onto a Chromolith CapRod column (150 X 0.1 mm, Merck) using a 5 µl injector loop on a Tempo LC MALDI Spotting system (ABI-MDS/Sciex). Buffer C is 2% acetonitrile, 0.1% trifluoroacetic acid, and Buffer D is 98% acetonitrile, 0.1% trifluoroacetic acid.

The elution gradient is 95% C/ 5% D (2ul per minute flowrate from 0-3 min, then 2.5ul per minute from 3-8.1 min), 5% D→38% D (8.1-40 min), 38% D→80% D (41-44 min), 80% D→5% D (44-49 min) (initial conditions). Flow rate is 2.5 µl/min during the gradient, and an equal flow of MALDI matrix solution is added post-column (7 mg/ml recrystallized CHCA (a-cyano-hydroxycinnamic acid), 2 mg/ml ammonium phosphate, 0.1% trifluoroacetic acid, 80% acetonitrile). (Gradient used prior to 2008 was 100% C (0-4 min), 0→10% D (4-10 min), 10%→25% D (10-30 min), 25%→40% D (30-35 min), 40%→80% D (35-38 min), 80% D (38-42 min), 80%→0% D (42-43 min), 0% D (43-50 min), with a flow rate of 3.0 ul per minute with an equal flow of MALDI matrix solution.)

The combined eluant is automatically spotted onto a stainless steel MALDI target plate every 6 seconds (0.6 µl per spot), for a total of 370 spots per original SCX fraction.

Mass Spec analysis:

After sample spot drying above, thirteen calibrant spots (ABI 4700 Mix) are added to each plate manually. MALDI target plates (15 per experiment) are analyzed in a data-dependent manner on an ABI 4800 MALDI TOF-TOF.

As each plate is entered into the instrument, a plate calibration/ MS Default calibration update is performed, and then the MS/MS default calibration is updated. MS Spectra are then acquired from each sample spot using the newly updated default calibration, using 500 laser shots per spot, laser intensity 3200 (this can change somewhat with laser age and tuning). A plate-wide interpretation is then automatically performed, choosing the highest peak of each observed m/z value for subsequent MS/MS analysis.

Up to 2500 laser shots at laser power 4200 are accumulated for each MS/MS spectrum. When the MS and MS/MS spectra from all 15 plates in a sample set have been acquired, protein identification and quantitation are performed using either the Paragon algorithm as implemented in Protein Pilot 3.0 software (version 2.01 prior to July 2009, from ABI/MDS-Sciex), or GPS Explorer software (ABI) and Matrix Sciences Mascot algorithm version 2.1, in either case searching the spectra against either species-specific subsets (plus common contaminants) of the NCBInr database concatenated with a reversed "decoy" version of itself (in July 2009 we are using the July versions of these FASTA databases, obtained from  http://www.ncbi.nlm.nih.gov/sites/entrez?db=Taxonomy&cmd=search&term= , but we upload the latest database every 3-6 months). We occasionally the UniProt/SwissProt database plus decoy database (release 52.4 in December, 2007, concatenated with a reversed "decoy" version of itself in use in October 2008)).

For the predominantly used ProteinPilot analyses, the preset Thorough (iTRAQ or Identification) Search settings are used, and identifications must have a ProteinPilot Unused Score > 1.3 (>95% Confidence interval) in order to be accepted (Click HERE to download an Excel Spreadsheet of all the modifications that are considered by ProteinPilot). In addition, the only protein IDs accepted MUST have a "Local False Discovery Rate" estimation of no higher than 5%, as calculated from the slope of the accumulated Decoy database hits by the PSPEP (Proteomics System Performance Evaluation Pipeline ) program by Sean Seymour and colleagues (Tang, W.H., Shilov, I.V., and Seymour, S.L. A Non-linear Fitting Method for Determining Local False Discovery Rates from Decoy Database Searches, Journal of Proteome Research 2008 Sep;7(9):3661-7. Epub 2008 Aug 14.PMID: 18700793))(Click here for PDF explaining PSPEP further). Note that this Local or "Instantaneous" FDR estimate is much more stringent than p<0.05 or 95% confidence scores in Mascot, Sequest, ProteinPilot, or the aggregate False Discovery Rate estimations (number of Decoy database IDs/Total IDs at any chosen threshold score) commonly used in the literature, and combined with the ProGroup algorithm included in ProteinPilot gives a very conservative and fully MIAPE-compliant list of proteins identified (i.e., Mascot and other lists of  "Proteins ID'd at p<0.05" will produce more numerous "significant" IDs from the same data, but those larger lists are highly likely to contain many more False Positive IDs). For additional discussion of False Discovery Rates and their estimation, please see "Calculating False Discovery Rates". For iTRAQ and LC-MudPit experiments analysed with ProteinPilot, we recommend accepting all protein IDs with a LOCAL estimated FDR of 5% or lower.

For Mascot searches, parameters used are 50-100 PPM mass error tolerance for MS spectra, 0.4 Da MS/MS error tolerance, no missed cuts, fixed modifications of carbamidomethylation (and iTRAQ (lysine) and iTRAQ (NH-terminus) for iTRAQ experiments, and variable modifications of methionine oxidation and deamidation. Individual peptides have to be identified with an Ion Score Confidence Interval % of at least 90% in order to contribute to protein identifications and quantitation; protein identifications had to have a Total Ion Score Confidence Interval% of at least 95% to be considered significant.

For Mascot searches, you should never accept IDs with final score cutoffs low enough to produce a global false discovery rate of more than 5%, with more stringent score cutoffs more commonly used to keep the global false discovery rate below 1%-2% for Mascot searches (global false discovery rate (FDR) calculation based on 2x the number of identifications from the reversed decoy portion of the concatenated database at any score cutoff value).

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Last modified September 09, 2009 03:21 PM          This page has been viewed times since July 2007