Proteomics Chemistries and LC Separations

 

 

In addition to using classic gel spot densitometric (2D Gels) or fluorescence (DIGE Gel) measurements to find proteins whose levels differ between two or more samples, identification and quantitation of protein level changes can be done by using chemical isotope labeling methods. In such methods, one tags all proteins from different samples with identical chemical tags that differ only in their distribution of heavy and light (non-radioactive) isotopes of the same atoms, e.g., 1H vs. 2H, 12C vs 13C, 14N vs. 15N. Where possible, this can be done in living cells or animal models by feeding heavy vs. light isotope versions of various naturally incorporated amino acids (sometimes called SILAC methods); however, for many studies such pre-labeling is not possible. In such cases various chemical isotope incorporation methods are available. We previously performed many analyses with Isotope Coded Affinity Tag (ICAT) methods, and over the past 7 years (2006-2013) we have used Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) methods to perform many hundreds of large scale analyses of complex samples, including human serum samples, animal and plant cell lysates, and cell organelle preps. Such experiments typically identify and quantitate the relative amounts of 500-2500 proteins from 4-8 samples at once (see further details below).

View our iTRAQ Sample preparation protocols >>

Standard sample separations for iTRAQ experiments done in the Mass Spec Facility (after the iTRAQ reagent labeling step) are identical to those for an LC-MALDI MudPit experiment:

  1. Separation of the combined 4 or 8 iTRAQ-labeled samples offline into 15 strong cation exchange fractions, using a 4.6 X 250 mm PolySULFOETHYL Aspartamide Strong Cation exchange column (PolyLC, Columbia, MD) with an ammonium formate gradient in 20% acetonitrile. (Details of these protocols can be viewed at "2D LC Procedures Used")
  2. SpeedVac drying and resuspension in H2O of all fractions (3X) to remove all acetonitrile and ammonium formate.
  3. Separation of each SCX fraction on a 15 cm Reprosil C18 nanoflow column on an Eksigent ChipLC system, and direct injection into our ABSciex 5600 TripleTOF mass spectrometer (For MALDI analyses, separation of each SCX fraction on an LC-Tempo nanoflow separation and MALDI spotting system, using a Chromolith CapRod column C18 column (150 X 0.1 mm, Merck), into 370 MALDI spots on a stainless steel MALDI target plate, then adding 13 calibration spots to the same target plate).
  4. Instrument is calibrated with an injection of beta-gal digest standard before each sample injection (For MALDI analyses, update plate calibration and MS/MS default calibration for each plate as it goes into our Applied Biosystems 5800 MALDI TOF TOF mass spectrometer).
  5. A 250 ms parent scan is acquired, then up to 50 MS/MS spectra over 2.5 seconds, then repeated with the next LC eluant fraction. (For MALDI analyses, acquisition of 400 laser shots for MS spectra from each spot, then data-dependent acquisition of MS/MS spectra for each peptide mass, with the MS/MS spectra taken from the spot containing the largest MS peak representing each of the peptide peaks observed across the entire plate).
  6. Combination of the MS/MS data from all 15 SCX fractions for a Paragon algorithm search (ProteinPilot 4.5 Software as of Jan 2013, against a concatenated normal and reversed database (SwissProt, NCBI, etc.). This search of a concatenated normal and reversed database allows estimation of the False Discovery Rate (or False Positive Rate) so that one can set the score limits for "positive IDs" to only accept IDs where the local False Discovery Rate estimate of the lowest ranking protein is 5%, and all higher ranking proteins have decreasingly lower probabilities of being false positives (See "Calculating False Discovery Rates" for more information on the importance of using False Discovery Rate estimations and how they are calculated.
  7. The resulting ProteinPilot .group files can be viewed with a Windows XP or later computer by installing a trial version of the ProteinPilot Software from http://www.absciex.com/products/software/proteinpilot-software - once the trial period is over, you can continue to use the software as a viewer for .group files produced by our licensed versions of the software.