Rigorous Determination of the Stoichiometry of Protein Phosphorylation Using Mass Spectrometry

Quantification of the stoichiometry of phosphorylation is usually achieved using a mixture of phosphatase treatment and differential isotopic labeling. Here, we introduce a new approach to the concomitant determination of absolute protein concentration and the stoichiometry of phosphorylation at predefined sites. The method exploits QconCAT to quantify levels of phosphorylated and nonphosphorylated peptide sequences in a phosphoprotein. The nonphosphorylated sequence is used to determine the absolute protein quantity and serves as a reference to calculate the extent of phosphorylation at the second peptide. Thus, the stoichiometry of phosphorylation and the absolute protein concentration can be determined accurately in a single experiment

Evaluation of dimethyl sulfoxide (DMSO) as a mobile phase additive during top 3 label-free quantitative proteomics

AbstractDimethyl sulfoxide (DMSO) has been advocated as a beneficial additive to electrospray solvents for peptide analysis due to the improved ionisation efficiency conferred. Previous reports have shown that the resultant improvements in peptide ion signal intensities are non-uniform. As a result, it was hypothesised that inclusion of DMSO in electrospray solvents could be detrimental to the outcome of intensity-based label-free absolute quantification approaches, specifically the top 3 method. The effect of DMSO as a mobile phase additive in top 3 label-free quantification was therefore evaluated. We show that inclusion of DMSO enhances data quality, improving the precision and number of proteins quantified, with no significant change to the quantification values observed in its absence

Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic C-Terminal Residue

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H(3)PO(4)), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H(3)PO(4) elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H(3)PO(4) upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13361-013-0770-2) contains supplementary material, which is available to authorized users

The use of selected reaction monitoring in quantitative proteomics

Selected reaction monitoring (SRM) has a long history of use in the area of quantitative MS. In recent years, the approach has seen increased application to quantitative proteomics, facilitating multiplexed relative and absolute quantification studies in a variety of organisms. This article discusses SRM, after introducing the context of quantitative proteomics (specifically primarily absolute quantification) where it finds most application, and considers topics such as the theory and advantages of SRM, the selection of peptide surrogates for protein quantification, the design of optimal SRM co-ordinates and the handling of SRM data. A number of published studies are also discussed to demonstrate the impact that SRM has had on the field of quantitative proteomics. </jats:p

Peptide scrambling during collision-induced dissociation is influenced by N-terminal residue basicity

‘Bottom up’ proteomic studies typically use tandem mass spectrometry data to infer peptide ion sequence, enabling identification of the protein whence they derive. The majority of such studies employ collision-induced dissociation (CID) to induce fragmentation of the peptide structure giving diagnostic b-, y-, and a- ions. Recently, rearrangement processes that result in scrambling of the original peptide sequence during CID have been reported for these ions. Such processes have the potential to adversely affect ion accounting (and thus scores from automated search algorithms) in tandem mass spectra, and in extreme cases could lead to false peptide identification. Here, analysis of peptide species produced by Lys-N proteolysis of standard proteins is performed and sequences that exhibit such rearrangement processes identified. The effect of increasing the gas-phase basicity of the N-terminal lysine residue through derivatization to homoarginine toward such sequence scrambling is then assessed. The presence of a highly basic homoarginine (or arginine) residue at the N-terminus is found to disfavor/inhibit sequence scrambling with a coincident increase in the formation of b(n-1)+H2O product ions. Finally, further analysis of a sequence produced by Lys-C proteolysis provides evidence toward a potential mechanism for the apparent inhibition of sequence scrambling during resonance excitation CID

The identification and characterisation of the target proteins of the anti-epileptic drug R-lacosamide

(2R)-N-Benzyl-2-acetamido-3-methoxypropionamide (lacosamide) is an anticonvulsant (Choi et al., 1996); under the brand name "Vimpat" this small molecule has recently been approved by the European Medicines Agency and the U.S. Food and Drug Administration for the treatment of epilepsy. The purpose of the research reported here is to develop and apply mass spectrometry approaches to the determination of protein targets of this novel therapeutic. The general strategy involves selecting potential target proteins using lacosamide analogues incorporating an 'affinity bait' to enable covalent modification binding to target proteins, and a 'chemical reporter' for the selective recovery of modified proteins. Lacosamide analogues are incubated with biological samples (primarily mouse brain extracts) and the modified proteins are recovered by introduction of a biotin tag (via the chemical reporter group). Streptavidin affinity chromatography is then used to enrich for bound molecules. The enriched proteins are subjected to tryptic digestion and the resultant peptides analysed by reversed phase liquid chromatography coupled with tandem MS, enabling recognition of proteins via database searching. Firstly, mass spectrometric characterisation of the biotinyl (R)-lacosamide analogue bound to model compounds was performed. Adducts with protected lysine, neurotensin and enolase were analysed. The data showed that ESI was more suitable for ionisation of modified peptides and proteins than MALDI. The biotin enrichment strategy was applied to mouse brain lysate to identify putative candidate target proteins. Twenty-eight candidate target proteins were identified. Moreover, the 14-3-3 protein family, CRMP2 and the sodium/potassium-transporting ATPase family showed preference for the biologically active(R)- isomer over the (S)- lacosamide analogue using a fluorescence tag. Three more biotinyl lacosamide analogues containing different affinity baits were used to enrich candidate target proteins of lacosamide. Most of the identified target proteins supported the findings of the analogue A. To indicate the binding sites, a method was developed for enriching peptides modified by the biotinyl (R)-lacosamide analogue, using streptavidin beads and subsequently analysed these biotinylated peptides using CID and ETD fragmentation methods. Neither fragmentation technique was optimal for elucidation of the sequence or site of modification of unknown target peptides. Purified recombinant proteins were therefore adducted with an AB-(R)-lacosamide analogue lacking the biotinprobe. This smaller (R)-lacosamide analogue underwent less fragmentation than the biotin analogue during CID and could be used for sequence and site identification of the modified peptides. In summary, the studies illustrated the power of MS to study drug mechanisms via the discovery of candidate protein targets.EThOS - Electronic Theses Online ServiceThe Royal Thai GovernmentGBUnited Kingdo

Profiling the Human Phosphoproteome to Estimate the True Extent of Protein Phosphorylation

Mass spectrometry-based phosphoproteomics allows large-scale generation of phosphorylation site data. However, analytical pipelines need to be carefully designed and optimised to minimise incorrect identification of phosphopeptide sequences or wrong localisation of phosphorylation sites within those peptides. Public databases such as PhosphoSitePlus (PSP) and PeptideAtlas (PA) compile results from published papers or openly available MS data, but to our knowledge, there is no database-level control for false discovery of sites, subsequently leading to the likely overestimation of true phosphosites. It is therefore difficult for researchers to assess which phosphosites are “real” and which are likely to be artefacts of data processing. By profiling the human phosphoproteome, we aimed to estimate the false discovery rate (FDR) of phosphosites based on available evidence in PSP and/or PA and predict a more realistic count of true phosphosites. We ranked sites into phosphorylation likelihood sets based on layers of accumulated evidence and then analysed them in terms of amino acid conservation across 100 species, sequence properties and functional annotations of associated proteins. We demonstrated significant differences between the sets and developed a method for independent phosphosite FDR estimation. Remarkably, we estimated a false discovery rate of 86.1%, 95.4% and 82.2% within sets of described phosphoserine (pSer), phosphothreonine (pThr) and phosphotyrosine (pTyr) sites respectively for which only a single piece of identification evidence is available (the vast majority of sites in PSP). Overall, we estimate that ∼56,000 Ser, 10,000 Thr and 12,000 Tyr phosphosites in the human proteome have truly been identified to date, based on evidence in PSP and/or PA, which is lower than most published estimates. Furthermore, our analysis estimated ∼91,000 Ser, 49,000 Thr and 26,000 Tyr sites that are likely to represent false-positive phosphosite identifications. We conclude that researchers should be aware of the significant potential for false positive sites to be present in public databases and should evaluate the evidence behind the phosphosites used in their research

RePLiCal: A QconCAT Protein for Retention Time Standardization in Proteomics Studies

This study introduces a new reversed-phase liquid chromatography retention time (RT) standard, RePLiCal (<b>Re</b>versed-<b>p</b>hase <b>li</b>quid chromatography <b>cal</b>ibrant), produced using QconCAT technology. The synthetic protein contains 27 lysine-terminating calibrant peptides, meaning that the same complement of standards can be generated using either Lys-C or trypsin-based digestion protocols. RePLiCal was designed such that each constituent peptide is unique with respect to all eukaryotic proteomes, thereby enabling integration into a wide range of proteomic analyses. RePLiCal has been benchmarked against three commercially available peptide RT standard kits and outperforms all in terms of LC gradient coverage. RePLiCal also provides a higher number of calibrant points for chromatographic retention time standardization and normalization. The standard provides stable RTs over long analysis times and can be readily transferred between different LC gradients and nUHPLC instruments. Moreover, RePLiCal can be used to predict RTs for other peptides in a timely manner. Furthermore, it is shown that RePLiCal can be used effectively to evaluate trapping column performance for nUHPLC instruments using trap-elute configurations, to optimize gradients to maximize peptide and protein identification rates, and to recalibrate the <i>m</i>/<i>z</i> scale of mass spectrometry data post-acquisition

cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility-mass spectrometry

cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro. We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R(2)C(2) holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a ‘kinase-dead’ PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM–MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling

Formation of carbohydrate-functionalised polystyrene and glass slides and their analysis by MALDI-TOF MS

Glycans functionalised with hydrophobic trityl groups were synthesised and adsorbed onto polystyrene and glass slides in an array format. The adsorbed glycans could be analysed directly on these minimally conducting surfaces by MALDI-TOF mass spectrometry analysis after aluminium tape was attached to the underside of the slides. Furthermore, the trityl group appeared to act as an internal matrix and no additional matrix was necessary for the MS analysis. Thus, trityl groups can be used as simple hydrophobic, noncovalently linked anchors for ligands on surfaces and at the same time facilitate the in situ mass spectrometric analysis of such ligands