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PAGE gel, individual proteins were either visualized by Coomassie Brilliant Blue stain excised and subjected to an in-gel tryptic digest (digests were performed as described (Shevchenko et al, 1996), or the eluted CenH3 particles were precipitated with 10% trichloroacetic acid, washed once with acetone, dried, re-dissolved in de-ionized water, and digested in solution with Arg-C protease. For CenH3 identification, we could not detect peptides from the protein directly in the gel slice containing the tetramer (confirmed by Western blot analysis). Therefore, we used eluted CenH3 protein from extensively washed CenH3IP-beads. Following digestion, samples were desalted using a microC18 ZipTip (Millipore) and dried. Samples were then resuspended, in 5 µL of 0.1% formic acid and analyzed by LC/ESI MS/MS (Liquid Chromatography coupled to Electrospray Ionization tandem mass spectrometry) with a nano2D LC (Eksigent) coupled to an LTQ-FT mass spectrometer (ThermoElectron) using an instrument configuration as described (Yi et al, 2003). Data were collected in a data-dependent mode in which a high mass resolution/high mass accuracy scan (in the FT part of the instrument) was followed by MS/MS scans of the five most abundant ions from the preceding MS scan (in the LTQ part of the instrument). The five selected ions for tandem MS were placed on an exclusion list and not selected for subsequent tandem mass spectrometry for 1.5 minutes, allowing less intense ions to be interrogated for tandem mass spectrometry. Proteins were identified from mass spectrometry data using a modified version of the open source X!Tandem (Beavis Informatics) automated protein database search algorithm. The score function of native X!Tandem was replaced with a dot-product based score algorithm that is compatible with Peptide Prophet (Keller et al, 2005). Search results were analyzed by the Peptide Prophet program and identifications were