Henk van den Toorn

Toward Full Peptide Sequence Coverage by Dual Fragmentation Combining Electron-Transfer and Higher-Energy Collision Dissociation Tandem Mass Spectrometry

Increasing peptide sequence coverage by tandem mass spectrometry improves confidence in database search-based peptide identification and facilitates mapping of post-translational modifications and de novo sequencing. Inducing 2-fold fragmentation by combining electron-transfer and higher-energy collision dissociation (EThcD) generates dual fragment ion series and facilitates extensive peptide backbone fragmentation. After an initial electron-transfer dissociation step, all ions including the unreacted precursor ions are subjected to collision induced dissociation which yields b/y- and c/z-type fragment ions in a single spectrum. This new fragmentation scheme provides richer spectra and substantially increases the peptide sequence coverage and confidence in peptide identification.

Quantitative and Qualitative Proteome Characteristics Extracted from In-Depth Integrated Genomics and Proteomics Analysis

Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and posttranscriptional levels. Here, we integrated in-depth transcriptome and proteome analyses of liver tissues from two rat strains to unravel the interactions within and between these layers. We obtained peptide evidence for 26,463 rat liver proteins. We validated 1,195 gene predictions, 83 splice events, 126 proteins with nonsynonymous variants, and 20 isoforms with nonsynonymous RNA editing. Quantitative RNA sequencing and proteomics data correlate highly between strains but poorly among each other, indicating extensive nongenetic regulation. Our multilevel analysis identified a genomic variant in the promoter of the most differentially expressed gene Cyp17a1, a previously reported top hit in genome-wide association studies for human hypertension, as a potential contributor to the hypertension phenotype in SHR rats. These results demonstrate the power of and need for integrative analysis for understanding genetic control of molecular dynamics and phenotypic diversity in a system-wide manner.

The PRoteomics IDEntification (PRIDE) Converter 2 Framework: An Improved Suite of Tools to Facilitate Data Submission to the PRIDE Database and the ProteomeXchange Consortium

The original PRIDE Converter tool greatly simplified the process of submitting mass spectrometry (MS)-based proteomics data to the PRIDE database. However, after much user feedback, it was noted that the tool had some limitations and could not handle several user requirements that were now becoming commonplace. This prompted us to design and implement a whole new suite of tools that would build on the successes of the original PRIDE Converter and allow users to generate submission-ready, well-annotated PRIDE XML files. The PRIDE Converter 2 tool suite allows users to convert search result files into PRIDE XML (the format needed for performing submissions to the PRIDE database), generate mzTab skeleton files that can be used as a basis to submit quantitative and gel-based MS data, and post-process PRIDE XML files by filtering out contaminants and empty spectra, or by merging several PRIDE XML files together. All the tools have both a graphical user interface that provides a dialog-based, user-friendly way to convert and prepare files for submission, as well as a command-line interface that can be used to integrate the tools into existing or novel pipelines, for batch processing and power users. The PRIDE Converter 2 tool suite will thus become a cornerstone in the submission process to PRIDE and, by extension, to the ProteomeXchange consortium of MS-proteomics data repositories.

An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas

Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti(4+)-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substantially and confirms the considerable tryptic bias in public repositories. We define and make available a less biased human phosphopeptide atlas of 37,771 unique phosphopeptides, correlating to 18,430 unique phosphosites, of which fewer than 1/3 were identified in more than one protease data set. We demonstrate that each protein phosphorylation site can be linked to a preferred protease, enhancing its detection by mass spectrometry (MS). For specific sites, this approach increases their detectability by more than 1,000-fold.

Effect of Chemical Modifications on Peptide Fragmentation Behavior upon Electron Transfer Induced Dissociation

In proteomics, proteolytic peptides are often chemically modified to improve MS analysis, peptide identification, and/or to enable protein/peptide quantification. It is known that such chemical modifications can alter peptide fragmentation in collision induced dissociation MS/MS. Here, we investigated the fragmentation behavior of such chemically modified peptides in MS/MS using the relatively new activation method electron transfer dissociation (ETD). We generated proteolytic peptides using the proteases Lys-N and trypsin and compared the fragmentation behavior of the unlabeled peptides with that of their chemically modified cognates. We investigated the effect of several commonly used modification reactions, namely, guanidination, dimethylation, imidazolinylation, and nicotinylation (ICPL). Of these guanidination and imidazolinylation specifically target the epsilon-amino groups of lysine residues in the peptides, whereas dimethylation and nicotinylation modify both N-termini and epsilon-amino groups of lysine residues. Dimethylation, guanidination, and particularly imidazolinylation of doubly charged Lys-N peptides resulted in a significant increase in peptide sequence coverage, resulting in more reliable peptide identification using ETD. This may be rationalized by the increased basicity and resulting positive charge at the N-termini of these peptides. Nicotinylation of the peptides, on the other hand, severely suppressed backbone fragmentation, hampering the use of this label in ETD based analysis. Doubly charged C-terminal lysine containing tryptic peptides also resulted in an enhanced observation of a single type of fragment ion series when guanidinated or imidazolinylated. These labels would thus facilitate the use of de novo sequencing strategies based on ETD for both arginine and lysine containing tryptic peptides. Since isotopic analogues of the labeling reagents applied in this work are commercially available, one can combine quantitation with improved ETD based peptide sequencing for both Lys-N and trypsin digested samples.

qcML: An Exchange Format for Quality Control Metrics from Mass Spectrometry Experiments

Quality control is increasingly recognized as a crucial aspect of mass spectrometry based proteomics. Several recent papers discuss relevant parameters for quality control and present applications to extract these from the instrumental raw data. What has been missing, however, is a standard data exchange format for reporting these performance metrics. We therefore developed the qcML format, an XML-based standard that follows the design principles of the related mzML, mzIdentML, mzQuantML, and TraML standards from the HUPO-PSI (Proteomics Standards Initiative). In addition to the XML format, we also provide tools for the calculation of a wide range of quality metrics as well as a database format and interconversion tools, so that existing LIMS systems can easily add relational storage of the quality control data to their existing schema. We here describe the qcML specification, along with possible use cases and an illustrative example of the subsequent analysis possibilities. All information about qcML is available at http://code.google.com/p/qcml.

Selectivity in Enrichment of cAMP-dependent Protein Kinase Regulatory Subunits Type I and Type II and Their Interactors Using Modified cAMP Affinity Resins

cAMP regulates cellular functions primarily by activating PKA. The involvement of PKAs in various signaling pathways occurring simultaneously in different cellular compartments necessitates stringent spatial and temporal regulation. This specificity is largely achieved by binding of PKA to protein scaffolds, whereby a distinct group of proteins called A kinase anchoring proteins (AKAPs) play a dominant role. AKAPs are a diverse family of proteins that all bind via a small PKA binding domain to the regulatory subunits of PKA. The binding affinities between PKA and several AKAPs can be different for different isoforms of the regulatory subunits of PKA. Here we employ a combination of affinity chromatography and mass spectrometry-based quantitative proteomics to investigate specificity in PKA-AKAP interactions. Three different immobilized cAMP analogs were used to enrich for PKA and its interacting proteins from several systems; HEK293 and RCC10 cells and rat lung and testis tissues. Stable isotope labeling was used to confidently identify and differentially quantify target proteins and their preferential binding affinity for the three different cAMP analogs. We were able to enrich all four isoforms of the regulatory subunits of PKA and concomitantly identify more than 10 AKAPs. A selective enrichment of the PKA RI isoforms could be achieved; which allowed us to unravel which AKAPs bind preferentially to the RI or RII regulatory domains of PKA. Of the twelve AKAPs detected, seven preferentially bound to RII, whereas the remaining five displayed at least dual specificity with a potential preference for RI. For some of these AKAPs our data provide the first insights into their specificity.

StatQuant: a post-quantification analysis toolbox for improving quantitative mass spectrometry

MOTIVATION Mass spectrometric protein quantitation has emerged as a high-throughput tool to yield large amounts of data on peptide and protein abundances. Currently, differential abundance data can be calculated from peptide intensity ratios by several automated quantitation software packages available. There is, however, still a great need for additional processing to validate and refine the quantitation results. Here, we present a software tool, termed StatQuant, that offers a set of statistical tools to process, filter, compare and represent data from several quantitative proteomics software packages such as MSQuant. StatQuant offers the researcher post-processing methods to achieve improved confidence on the obtained protein ratios. AVAILABILITY StatQuant can be downloaded from: (https://gforge.nbic.nl/projects/statquant/) (binary and source code).

Deep proteome profiling of Trichoplax adhaerens reveals remarkable features at the origin of metazoan multicellularity

Genome sequencing of arguably the simplest known animal, Trichoplax adhaerens, uncovered a rich array of transcription factor and signalling pathway genes. Although the existence of such genes allows speculation about the presence of complex regulatory events, it does not reveal the level of actual protein expression and functionalization through posttranslational modifications. Using high-resolution mass spectrometry, we here semi-quantify 6,516 predicted proteins, revealing evidence of horizontal gene transfer and the presence at the protein level of nodes important in animal signalling pathways. Moreover, our data demonstrate a remarkably high activity of tyrosine phosphorylation, in line with the hypothesized burst of tyrosine-regulated signalling at the instance of animal multicellularity. Together, this Trichoplax proteomics data set offers significant new insight into the mechanisms underlying the emergence of metazoan multicellularity and provides a resource for interested researchers.

RockerBox: Analysis and Filtering of Massive Proteomics Search Results

A major problem in the analysis of mass spectrometry-based proteomics data is the vast growth of data volume, caused by improvements in sequencing speed of mass spectrometers. This growth affects analysis times and storage requirements so severely that many analysis tools are no longer able to cope with the increased file sizes. We present a tool, RockerBox, to address size problems for search results obtained from the widely used Mascot search engine. RockerBox allows for a fast evaluation of large result files by means of a number of commonly accepted metrics that can often be viewed through charts. Moreover, result files can be filtered without altering their informative content, based on a number of FDR calculation methods. File sizes can be reduced dramatically, often to a tenth of their original size, thus relaxing the need for storage and computation power, and boosting analysis of current and future proteomics experiments.