Joerg Seidler

De novo sequencing of peptides by MS/MS

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 18O labeling, MSn 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.

GCP6 is a substrate of Plk4 and required for centriole duplication

Centriole 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 γ-tubulin ring complex (γ-TuRC) that nucleates microtubules. GCP6 is a member of the γ-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 γ-TuRC and induces a high percentage of monopolar spindles. These spindles are characterized by a loss of centrosomal γ-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.

Citrate boosts the performance of phosphopeptide analysis by UPLC-ESI-MS/MS.

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.

Toward Comprehensive Analysis of the Galectin Network in Chicken: Unique Diversity of Galectin-3 and Comparison of its Localization Profile in Organs of Adult Animals to the Other Four Members of this Lectin Family

Characterization of all members of a gene family established by gene divergence is essential to delineate distinct or overlapping expression profiles and functionalities. Their activity as potent modulators of diverse physiological processes directs interest to galectins (endogenous lectins with β‐sandwich fold binding β‐galactosides and peptide motifs), warranting their study with the long‐term aim of a comprehensive analysis. The comparatively low level of complexity of the galectin network in chicken with five members explains the choice of this organism as model. Previously, the three proto‐type chicken galectins CG‐1A, CG‐1B, and CG‐2 as well as the tandem‐repeat‐type CG‐8 had been analyzed. Our study fills the remaining gap to determine gene structure, protein characteristics and expression profile of the fifth protein, that is, chimera‐type chicken galectin‐3 (CG‐3). Its gene has a unique potential to generate variants: mRNA production stems from two promoters, alternative splicing of the form from the second transcription start point (tsp) can generate three mRNAs. The protein with functional phosphorylation sites in the N‐terminus generated by transcription from the first tsp (tsp1CG‐3) is the predominant CG‐3 type present in adult tissues. Binding assays with neoglycoproteins and cultured cells disclose marked similarity to properties of human galectin‐3. The expression and localization profiles as well as proximal promoter regions have characteristic features distinct from the other four CGs. This information on CG‐3 completes the description of the panel of CGs, hereby setting the stage for detailed comparative analysis of the entire CG family, e.g., in embryogenesis. Anat Rec, , 2011.

Analysis of autophosphorylation sites in the recombinant catalytic subunit alpha of cAMP-dependent kinase by nano-UPLC-ESI-MS/MS

AbstractThe catalytic subunit of recombinant wild-type cyclic adenosine monophosphate-dependent protein kinase A (PKA) has been analyzed by a combination of 1D gel electrophoresis, in-gel digestion by trypsin, chymotrypsin, or endoproteinase AspN, and nano-ultraperformance liquid chromatography–MS/MS. The MS/MS spectra were annotated by MASCOT and the annotations were manually controlled. Using Ga(III)-immobilized metal ion affinity chromatography (IMAC), in addition to the four established autophosphorylation sites of the catalytic subunit of recombinant PKA, pSer10, pSer139, pThr197, and pSer338, six new phosphorylated residues have been characterized–pSer14, pThr48, pSer53, pSer212, pSer259, and pSer325. The established phosphorylation sites all are part of a PKA consensus motif and were found to be almost completely modified. In contrast, the newly detected sites were only partially phosphorylated. For estimation of their degree of phosphorylation, a method based on signal intensity measurements was used. For this purpose, signal intensities of all phospho- and non-phosphopeptides containing a particular site were added for estimation of site-specific phosphorylation degrees. This addition was performed over all peptides observed in the different digestion experiments, including their different charge states. pThr48 and pSer259 are located within PKA consensus motifs and were observed to be phosphorylated at 20% and 24%, respectively. pSer14 and pSer53 are located within inverted PKA consensus motifs and were found to be phosphorylated around 10% and 15%, respectively. The sequence environments of pSer212 and pSer325 have no similarity to the PKA consensus motif at all and were observed to be phosphorylated at about 5% or lower. All newly observed phosphorylation sites are located at the surface of the native protein structure of the PKA catalytic subunit. The results add new information on the theme of site-specific (auto)phosphorylation by PKA. FigureSequence of protein kinase A Ca (P00517) with highlighted autophosphorylation sites as determined by UPLC-MS/MS. The 4 known autophosphorylation sites are printed in blue and the 6 newly detected autophosphorylation are printed in red.

Protein phosphorylation influences proteolytic cleavage and kinase substrate properties exemplified by analysis of in vitro phosphorylated Plasmodium falciparum glideosome-associated protein 45 by nano-ultra performance liquid chromatography–tandem mass spectrometry

Plasmodium falciparum glideosome-associated protein 45 (PfGAP45) was in vitro phosphorylated by P. falciparum calcium-dependent protein kinase (PfCDPK1) and digested using the four proteases trypsin, chymotrypsin, AspN, and elastase. Subsequently, phosphopeptide enrichment using Ga(III) immobilized metal affinity chromatography (IMAC) was performed. The resulting fractions were analyzed using ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), resulting in the identification of a total of nine phosphorylation sites: Ser31, Ser89, Ser103, Ser109, Ser121, Ser149, Ser156, Thr158, and Ser173. During in-depth analyses of the detected phosphopeptides, it was observed that phosphorylation alters the properties of PfGAP45 as kinase and protease substrate. The closely adjacent phosphorylation sites Ser156 (major site) and Thr158 (minor site) were analyzed in detail because at first glance the specific proteases gave highly variable results with respect to the relative abundance of these sites. It was observed that (i) formation of pSer156 and pThr158 was mutually exclusive and (ii) phosphorylation at Ser156 or Thr158 interfered specifically with proteolysis by chymotrypsin or trypsin, respectively. The latter effect was studied in detail using synthetic phosphopeptides carrying either pSer156 or pThr158 as substrate for chymotrypsin or trypsin, respectively.

Minimally permutated peptide analogs as internal standards for relative and absolute quantification of peptides and proteins

A novel type of isobaric internal peptide standard for quantitative proteomics is described. The standard is a synthetic peptide derived from the target peptide by positional permutation of two amino acids. This type of internal standard is denominated minimally permutated peptide analog (MIPA). MIPA can be differentiated from their target analytes by LC‐MS due to individual retention times and/or by MS/MS due to specific fragment ions. Both quantification methods are demonstrated using peptide mixtures of low and high complexity.

Site-specific degree of phosphorylation in proteins measured by liquid chromatography-electrospray mass spectrometry

This review focuses on quantitative protein phosphorylation analysis based on coverage of both the phosphorylated and nonphosphorylated forms. In this way, site‐specific data on the degree of phosphorylation can be measured, generating the most detailed level of phosphorylation status analysis of proteins. To highlight the experimental challenges in this type of quantitative protein phosphorylation analysis, we discuss the typical workflows for mass spectrometry‐based proteomics with a focus on the quantitative analysis of peptide/phosphopeptide ratios. We review workflows for measuring site‐specific degrees of phosphorylation including the label‐free approach, differential stable isotope labeling of analytes, and methods based on the addition of stable isotope labeled peptide/phosphopeptide pairs as internal standards. The discussion also includes the determination of phosphopeptide isoform abundance data for multiply phosphorylated motifs that contain information about the connectivity of phosphorylation events. The review closes with a prospective on the use of intact stable isotope labeled proteins as internal standards and a summarizing discussion of the typical accuracies of the individual methods.

Structural and mechanistic information on c1 ion formation in collision-induced fragmentation of peptides

The formation of c1 ions during collision-induced fragmentation of peptides with asparagine, ornithine, or glutamine at the N-terminal position 2 has been studied. For this purpose, the corresponding fragment ion spectra of a large set of synthetic peptides were investigated. It is demonstrated that the c1 ion intensity depends on the nature of the second residue in the N-terminal dipeptide motif as well as on the peptide length. It is shown that the formation of c1 ions proceeds by two competing mechanisms. One mechanism is the secondary fragmentation of the b2 ion, the efficiency of which shows only a minor dependency on the complete peptide sequence. The other mechanism is the direct formation from the molecular ion, which is identified to be connected with sequence-specific c1 ion intensities. A model for this latter mechanism is proposed based on the analysis of the formation and secondary fragmentation of the zmax-1 ion, which is the complementary ion to the c1 ion. Additional evidence is obtained by investigation of peptides with ornithine in N-terminal position 2, which in general exhibit c1 ion intensities intermediate between the asparagine- and glutamine-containing species. The data presented support the reliable assignment of N-terminal dipeptide motifs using collision-induced dissociation.

Defining the structural requirements for ribose 5-phosphate-binding and intersubunit cross-talk of the malarial pyridoxal 5-phosphate synthase

MINT‐7994425, MINT‐7994413, MINT‐7994435: PfPdx1 (uniprotkb:C6KT50) and PfPdx1 (uniprotkb:C6KT50) bind (MI:0407) by cosedimentation in solution (MI:0028).