Reliable characterization of post-translational modifications (PTMs) by mass spectrometry is a formidable challenge. The Proteomics Standards Research Group (sPRG) has been conducting a study that focuses on generation of a standard that can be used for assessment of existing methods and development of new approaches for detection of an array of PTMs in a complex proteome. The study sample contained a mixture of 70 synthetic peptides representing a variety of modifications, including acetylation, methylation, nitration, phosphorylation, and sulfation. The synthetic peptides were provided as two mixtures: peptides alone, and peptides mixed with a tryptic digest of six proteins from which the sequences of the synthetic peptides were derived. The individual proteins were purified, digested and analyzed prior to formulation of the sample. The synthetic peptides were characterized individually and then mixed together (with or without the digest) in specified quantities. The samples were comprehensively analyzed by the sPRG using a variety of instrumental and bioinformatics approaches, followed by construction of a spectral library. In addition, the stability of the sample constituents was evaluated using different storage conditions over a three-month period. The final samples were supplied to study participants in sufficient quantities to permit evaluation by several analytical approaches. The presentation will include an overview of the various analytical approaches used for detection and localization of PTMs, including choices in separation, instrumentation, ion fragmentation, and informatics. Summaries will be provided of the results acquired by the sPRG members as well as the responses from participants who completed the study. Other highlights of the presentation include discussions about the modified peptides that were most difficult to detect, the results of the sample stability study, and a comparison of a spectral library-based peptide identification approach versus traditional database searching. Finally, proposed compositions of a standard planned for future commercialization will be considered.