Currently data-dependent acquisition (DDA) is the method of choice for mass spectrometry-based proteomics discovery experiments, but data-independent acquisition (DIA) is steadily becoming more important. One of the most important requirements to perform a DIA analysis is the availability of suitable spectral libraries for peptide identification and quantification. Several studies were performed addressing the evaluation of spectral library performance for protein identification in DIA measurements. But so far only few experiments estimate the effect of these libraries on the quantitative level. In this work we created a gold standard spike-in sample set with known contents and ratios of proteins in a complex protein matrix that allowed a detailed comparison of DIA quantification data obtained with different spectral library approaches. We used in-house generated sample-specific spectral libraries created using varying sample preparation approaches and repeated DDA measurement. In addition, two different search engines were tested for protein identification from DDA data and subsequent library generation. In total, eight different spectral libraries were generated, and the quantification results compared with a library free method, as well as a default DDA analysis. Not only the number of identifications on peptide and protein level in the spectral libraries and the corresponding DIA analysis results was inspected, but also the number of expected and identified differentially abundant protein groups and their ratios. We found, that while libraries of prefractionated samples were generally larger, there was no significant increase in DIA identifications compared with repetitive non-fractionated measurements. Furthermore, we show that the accuracy of the quantification is strongly dependent on the applied spectral library and whether the quantification is based on peptide or protein level. Overall, the reproducibility and accuracy of DIA quantification is superior to DDA in all applied approaches. Data has been deposited to the ProteomeXchange repository with identifiers PXD012986, PXD012987, PXD012988 and PXD014956.

The amount of public proteomics data is rapidly increasing but there is no standardized format to describe the sample metadata and their relationship with the dataset files in a way that fully supports their understanding or reanalysis. Here we propose to develop the transcriptomics data format MAGE-TAB into a standard representation for proteomics sample metadata. We implement MAGE-TAB-Proteomics in a crowdsourcing project to manually curate over 200 public datasets. We also describe tools and libraries to validate and submit sample metadata-related information to the PRIDE repository. We expect that these developments will improve the reproducibility and facilitate the reanalysis and integration of public proteomics datasets.

Controlled vocabularies (CVs), i.e. a collection of predefined terms describing a modeling domain, used for the semantic annotation of data, and ontologies are used in structured data formats and databases to avoid inconsistencies in annotation, to have a unique (and preferably short) accession number and to give researchers and computer algorithms the possibility for more expressive semantic annotation of data. The Human Proteome Organization (HUPO)–Proteomics Standards Initiative (PSI) makes extensive use of ontologies/CVs in their data formats. The PSI-Mass Spectrometry (MS) CV contains all the terms used in the PSI MS–related data standards. The CV contains a logical hierarchical structure to ensure ease of maintenance and the development of software that makes use of complex semantics. The CV contains terms required for a complete description of an MS analysis pipeline used in proteomics, including sample labeling, digestion enzymes, instrumentation parts and parameters, software used for identification and quantification of peptides/proteins and the parameters and scores used to determine their significance. Owing to the range of topics covered by the CV, collaborative development across several PSI working groups, including proteomics research groups, instrument manufacturers and software vendors, was necessary. In this article, we describe the overall structure of the CV, the process by which it has been developed and is maintained and the dependencies on other ontologies. Database URL: http://psidev.cvs.sourceforge.net/viewvc/psidev/psi/psi-ms/mzML/controlledVocabulary/psi-ms.obo

Protein inference connects the peptide spectrum matches (PSMs) obtained from database search engines back to proteins, which are typically at the heart of most proteomics studies. Different search engines yield different PSMs and thus different protein lists. Analysis of results from one or multiple search engines is often hampered by different data exchange formats and lack of convenient and intuitive user interfaces. We present PIA, a flexible software suite for combining PSMs from different search engine runs and turning these into consistent results. PIA can be integrated into proteomics data analysis workflows in several ways. A user-friendly graphical user interface can be run either locally or (e.g., for larger core facilities) from a central server. For automated data processing, stand-alone tools are available. PIA implements several established protein inference algorithms and can combine results from different search engines seamlessly. On several benchmark data sets, we show that PIA can identify a larger number of proteins at the same protein FDR when compared to that using inference based on a single search engine. PIA supports the majority of established search engines and data in the mzIdentML standard format. It is implemented in Java and freely available at https://github.com/mpc-bioinformatics/pia.

Cerebrospinal fluid is investigated in biomarker studies for various neurological disorders of the central nervous system due to its proximity to the brain. Currently, only a limited number of biomarkers have been validated in independent studies. The high variability in the protein composition and protein abundance of cerebrospinal fluid between as well as within individuals might be an important reason for this phenomenon. To evaluate this possibility, we investigated the inter- and intraindividual variability in the cerebrospinal fluid proteome globally, with a specific focus on disease biomarkers described in the literature. Cerebrospinal fluid from a longitudinal study group including 12 healthy control subjects was analyzed by label-free quantification (LFQ) via LC-MS/MS. Data were quantified via MaxQuant. Then, the intra- and interindividual variability and the reference change value were calculated for every protein. We identified and quantified 791 proteins, and 216 of these proteins were abundant in all samples and were selected for further analysis. For these proteins, we found an interindividual coefficient of variation of up to 101.5% and an intraindividual coefficient of variation of up to 29.3%. Remarkably, these values were comparably high for both proteins that were published as disease biomarkers and other proteins. Our results support the hypothesis that natural variability greatly impacts cerebrospinal fluid protein biomarkers because high variability can lead to unreliable results. Thus, we suggest controlling the variability of each protein to distinguish between good and bad biomarker candidates, e.g., by utilizing reference change values to improve the process of evaluating potential biomarkers in future studies.

This paper focuses on the use of controlled vocabularies (CVs) and ontologies especially in the area of proteomics, primarily related to the work of the Proteomics Standards Initiative (PSI). It describes the relevant proteomics standard formats and the ontologies used within them. Software and tools for working with these ontology files are also discussed. The article also examines the "mapping files" used to ensure correct controlled vocabulary terms that are placed within PSI standards and the fulfillment of the MIAPE (Minimum Information about a Proteomics Experiment) requirements. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.