Fig. 2.

Analysis of human nuclear phosphorylation sites by multidimensional LC coupled to MS/MS/MS. (A) Eight milligrams of nuclear extract from asynchronous HeLa cells were separated by SDS/PAGE. The entire gel was excised into 10 regions and proteolyzed with trypsin followed by phosphopeptide enrichment by SCX LC. Early eluting fractions were subjected to amino acid sequence analysis by reverse-phase LC-MS/MS with data-dependent MS/MS/MS acquisition. Phosphorylation sites (n = 2,002) were identified by the sequest algorithm, acquisition of MS/MS/MS spectra, and manual validation. Prep, preparation. (B) Example of a MS/MS spectrum of a phosphopeptide showing a typical extensive neutral loss of phosphoric acid. (C)MS/MS/MS spectrum of the neutral loss precursor ion from B. Abundant peptide bond fragmentation permitted the unambiguous identification of this peptide from the protein cell division cycle 2-related protein kinase 7, with a phosphorylated Ser residue marked by an asterisk.