The current status of de novo sequencing of peptides by MS/MS is reviewed with focus on collision cell MS/MS spectra. The relation between peptide structure and observed fragment ion series is discussed and the exhaustive extraction of sequence information from CID spectra of protonated peptide ions is described. The partial redundancy of the extracted sequence information and a high mass accuracy are recognized as key parameters for dependable de novo sequencing by MS. In addition, the benefits of special techniques enhancing the generation of long uninterrupted fragment ion series for de novo peptide sequencing are highlighted. Among these are terminal 18 O labeling, MS n of sodiated peptide ions, N-terminal derivatization, the use of special proteases, and time-delayed fragmentation. The emerging electron transfer dissociation technique and the recent progress of MALDI techniques for intact protein sequencing are covered. Finally, the integration of bioinformatic tools into peptide de novo sequencing is demonstrated.

SummaryCentriole duplication occurs once per cell cycle and requires Plk4, a member of the Polo-like kinase family. A key component of the centrosome is the c-tubulin ring complex (c-TuRC) that nucleates microtubules. GCP6 is a member of the c-TuRC, but its role in human cells and the regulation of its functions remain unclear. Here we report that depletion of human GCP6 prevents assembly of the c-TuRC and induces a high percentage of monopolar spindles. These spindles are characterized by a loss of centrosomal c-tubulin and reduced centriole numbers. We found that GCP6 is localized in the pericentriolar material but also at distal portions of centrioles. In addition, GCP6 is required for centriole duplication and Plk4-induced centriole overduplication. GCP6 interacts with and is phosphorylated by Plk4. Moreover, we find that Plk4-dependent phosphorylation of GCP6 regulates centriole duplication. These data suggest that GCP6 is a target of Plk4 in centriole biogenesis.

Incomplete recovery from the LC column is identified as a major cause for poor detection efficiency of phosphopeptides by LC-MS/MS. It is proposed that metal ions adsorbed on the stationary phase interact with the phosphate group of phosphopeptides via an ion-pairing mechanism related to IMAC (IMAC: immobilized metal ion affinity chromatography). This may result in their partial or even complete retention. Addition of phosphate, EDTA or citrate to the phosphopeptide sample was tested to overcome the detrimental phosphopeptide suppression during gradient LC-MS/MS analysis, while the standard solvent composition (water, acetonitrile, formic acid) of the LC system was left unchanged. With the use of UPLC, a citrate additive was found to be highly effective in increasing the phosphopeptide detection sensitivity. Addition of EDTA was found to be comparable with respect to sensitivity enhancement, but led to fast clogging and destruction of the spray needle and analytical columns due to precipitation. In contrast, a citrate additive is compatible with prolonged and stable routine operation. A 50 mM citrate additive was tested successfully for UPLC-MS analysis of a commercial four-component phosphopeptide mixture, a tryptic beta-casein digest, and several digests of the 140 kDa protein SETDB1. In this protein, 27 phosphorylation sites could be identified by UPLC-MS/MS using addition of citrate, including the detection of several phosphopeptides carrying 3-5 pSer/pThr residues, compared to identification of only 10 sites without citrate addition. A 50 mM citrate additive particularly increases the recovery of multiply phosphorylated peptides, thus, extending the scope of phosphopeptide analysis by LC-MS/MS.