Proteomics Standards Initiative Extended FASTA Format

Abstract

Mass-spectrometry-based proteomics enables the high-throughput identification and quantification of proteins, including sequence variants and post-translational modifications (PTMs) in biological samples. However, most workflows require that such variations be included in the search space used to analyze the data, and doing so remains challenging with most analysis tools. In order to facilitate the search for known sequence variants and PTMs, the Proteomics Standards Initiative (PSI) has designed and implemented the PSI Extended FASTA Format (PEFF). PEFF is based on the very popular FASTA format but adds a uniform mechanism for encoding substantially more metadata about the sequence collection as well as individual entries, including support for encoding known sequence variants, PTMs, and proteoforms. The format is very nearly backward compatible, and as such, existing FASTA parsers will require little or no changes to be able to read PEFF files as FASTA files, although without supporting any of the extra capabilities of PEFF. PEFF is defined by a full specification document, controlled vocabulary terms, a set of example files, software libraries, and a file validator. Popular software and resources are starting to support PEFF, including the sequence search engine Comet and the knowledge bases neXtProt and UniProtKB. Widespread implementation of PEFF is expected to further enable proteogenomics and top-down proteomics applications by providing a standardized mechanism for encoding protein sequences and their known variations. All the related documentation, including the detailed file format specification and example files, are available at http://www.psidev.info/peff.

UniProt: the universal protein knowledgebase in 2021

“…Peptide identifications can be taken as evidence that a protein has been validated (PE = 1) using a variation of the HPP guidelines (in brief, at least two unique peptides of seven amino acids or more or, for proteins where this cannot be achieved due to sequence constraints, one unique peptide of ten amino acids or more has been mapped to a protein). UniProt also provides the new format PEFF (PSI Extended FASTA Format) proposed by the HUPO-PSI (Human Proteome Organization-Proteomics Standard Initiative) for sequence databases ( 39 ) to be used by sequence search engines and other associated tools (e.g. spectral libraries search tools).…”

“…These include the website RESTful Application Programming Interface (API), stable URLs that can be bookmarked, linked, and reused, the Proteins extended REST API providing genomic coordinates of UniProtKB sequences and annotations imported and mapped from large-scale data imports ( 29 ), and the SPARQL API that allows users to perform complex queries across all UniProt data and also other resources that provide a SPARQL endpoint, such as DisGeNET ( 45 ), Bgee ( 46 ), or Wikidata ( 47 ). The Proteins API has recently been extended to serve the HUPO Proteomics Standards Initiative Extended FASTA Format (PEFF) for the proteomics community which enables more metadata, such as detail of amino-acid variants in the FASTA file header section ( 39 ).…”

Assessing Protein Sequence Database Suitability Using De Novo Sequencing

“…This topic is worth revisiting, given the availability and capabilities of Novor. A second option might employ an open modification search strategy to try to identify these novel sequences or modified peptides (24), and a third option would be to perform a search using an extended FASTA database (25).…”

What will neXtProt help us achieve in 2020 and beyond?

“…As for past releases, PTM and variant information included in the 2020-01-17 release are made available in various formats, including the HUPO PSI PEFF [12] that allows protein identification software to consider PTMs and variants in the search space when analyzing spectra. The peptide uniqueness checker already takes variants into account when mapping peptides to entries [9].…”