A total of 68,035 peptides from 5,558 reference proteins and 280 peptides from 129 alternative proteins were identified. The mean sequence coverage for reference and alternative proteins was 28.8% and 32.3%, respectively. Overall, alternative proteins represented 2.27% of the total identified proteins. This result clearly shows that the contribution of alternative proteins to the proteome, and thus the number of multiple coding genes, has been overlooked. It is noteworthy that alternative proteins coding sequences are spread across the different regions of mRNAs in agreement with the predicted distribution. Co-Tulathromycin B expression of an alternative protein and its reference protein was observed for 42 genes. For each of these genes, the average peptide intensity plot of both the reference and alternative proteins revealed large variations in co-expression ratio, indicating that a reference protein might not always be the main protein product of a gene. To confirm the expression of alternative proteins in cell lines different from HeLa cells, we performed LCMS/MS on human colon cell lines and identified 45 alternative proteins. AltORFs associated with these 45 proteins were distributed within UTRs and RefORFs with frequencies comparable to those observed in HeLa cells. Comparative analysis of alternative proteins detected in both HeLa cells and colon cell lines indicated that 14 are expressed in at least two cell lineages. This is more than expected by chance. SDS-PAGE in combination with LC-MS/MS is generally limited to the analysis of proteins above 10 kDa, and a low molecular weight is a known limitation in protein identification by MS. Since the majority of the predicted alternative proteome is composed of proteins less than 90 amino acids long which have a predicted molecular weight below 10 kDa, it is not surprising to have detected much more peptides corresponding to the conventional proteome compared to the alternative proteome. To further assess the abundance of the alternative proteome compared to the conventional proteome, HeLa cells proteins were separated by 1-D SDS-PAGE, and one gel slice between the 4.6 and 10 kDa markers was trypsin digested. The resulting peptides were analyzed by LC-MS/MS. A total of 44 reference and 14 alternative proteins were detected, and alternative proteins represented 24.14% of the total identified proteins, thus showing that alternative proteins are enriched in the pool of small cellular proteins. The detection of alternative proteins with MW between 4.78 and 9.49 kDa is further proof that peptides were not misassigned and that these alternative proteins are actually expressed. Next, we tested the expression of alternative proteins in a variety of human tissues by LC-MS/MS. First, we analyzed normal colon and lung tissues and detected 13 and 40 alternative proteins respectively. In a second set of experiments, we analyzed ovarian cancer tissue areas and normal areas from the same formalin fixed, paraffin-embedded tissue section of two patients, one presenting endometrioid ovarian cancer and the second presenting a serous ovarian cancer. A total of 19 alternative proteins were identified in the normal endometrium, endometrioid ovary, serous ovary, normal ovary, and serous fallopian tube. We completed these proteomic studies with human fluids, including cerebrospinal fluid, urine, plasma, and serum, identifying 16, 47, 90, and 928 alternative proteins in each fluid respectively. Strikingly, alternative proteins reButenafine hydrochloride present approximately 55% of the proteins identified in plasma and serum. Overall, we detected a total of 1,259 alternative proteins, and 47 were expressed in different cell lines and/or tissues. In accordance with the scanning model of translation initiation, we used the first AUG rule in order to predict the TIS of AltORFs present in our database.