The accidental or fraudulent blending of meat from different species is a highly relevant aspect for food product quality control, especially for consumers with ethical concerns against species, such as horse or pork. In this study, we present a sensitive mass spectrometrical approach for the detection of trace contaminations of horse meat and pork and demonstrate the specificity of the identified biomarker peptides against chicken, lamb, and beef. Biomarker peptides were identified by a shotgun proteomic approach using tryptic digests of protein extracts and were verified by the analysis of 21 different meat samples from the 5 species included in this study. For the most sensitive peptides, a multiple reaction monitoring (MRM) method was developed that allows for the detection of 0.55% horse or pork in a beef matrix. To enhance sensitivity, we applied MRM(3) experiments and were able to detect down to 0.13% pork contamination in beef. To the best of our knowledge, we present here the first rapid and sensitive mass spectrometrical method for the detection of horse and pork by use of MRM and MRM(3).

For the C-HPP consortium, dark proteins include not only uPE1, but also missing proteins (MPs, PE2-4), smORFs, proteins from lncRNAs, and products from uncharacterized transcripts. Here, we investigated the expression of dark proteins in the human testis by combining public mRNA and protein expression data for several tissues and performing LC-MS/MS analysis of testis protein extracts. Most uncharacterized proteins are highly expressed in the testis. Thirty could be identified in our data set, of which two were selected for further analyses: (1) A0AOU1RQG5, a putative cancer/testis antigen specifically expressed in the testis, where it accumulates in the cytoplasm of elongated spermatids; and (2) PNMA6E, which is enriched in the testis, where it is found in the germ cell nuclei during most stages of spermatogenesis. Both proteins are coded on Chromosome X. Finally, we studied the expression of other dark proteins, uPE1 and MPs, in a series of human tissues. Most were highly expressed in the testis at both the mRNA and protein levels. The testis appears to be a relevant organ to study the dark proteome, which may have a function related to spermatogenesis and germ cell differentiation. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium under the data set identifier PXD009598.

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