The data showed the potential and applicability of our immunoaffinity-based mass spectrometry GPS platform for reproducible quantitative proteomics targeting classes of motif-containing peptides. EXPERIMENTAL PROCEDURES Production of CIMS Antibodies Six human recombinant CIMS scFv antibodies (clones 1-B03, 15-A06, 17-E02, 32C3A-G03, 33C3D-F06, and 34C3A-D10) directed against six short C-terminal amino acid peptide motifs (denoted M-1, M-15, M17, M-32, M-33, and M-34), were selected from the n-CoDeR library (24), and kindly provided by BioInvent International AB, Lund, Sweden (supplemental Table S1). manner. On average, each of six different motif-specific antibodies was found to target about 75 different motif-containing proteins. Furthermore, peptides originating from proteins spanning in abundance from over a million down to less than 50 copies per cell, could be targeted. It is worth noting PA-824 (Pretomanid) that a significant set of peptides previously not reported in the PeptideAtlas database was PA-824 (Pretomanid) among the profiled targets. The quantitative data corroborated well with the corresponding data generated after conventional strong cation exchange fractionation of the same samples. Finally, several differentially expressed proteins, PA-824 (Pretomanid) with both known and unknown functions, many relevant for the central carbon metabolism, could be detected in the glucose- ethanol-cultivated yeast. Taken together, the study exhibited the potential of our immunoaffinity-based mass spectrometry platform for reproducible quantitative proteomics targeting classes of motif-containing peptides. In the quest for disease-associated biomarkers, the deciphering of the human proteome(s) will be central (1). Albeit powerful mass spectrometry (MS)-based technology platforms have been developed and frequently applied (2C4), the output in terms of validated biomarkers have so far been limited, mainly due to technological issues (5, PA-824 (Pretomanid) 6). In recent years, affinity proteomics based on antibody microarrays have become an established proteomic technology for protein expression profiling of complex proteomes (7C11). To date, the technology has been applied in several clinical applications, demonstrating its potential for, biomarker discovery, improved diagnosis and prognosis, as well as classification (8, 12C15). Despite the success, the possibility to run large-scale and, in particular, discovery mode projects using the conventional antibody array designs have been limited (7, 8, 16). First, antibodies of only known specificities, directed against preselected targets of a known specie, have been included, thereby excluding the possibility to discover novel targets (across species). Second, the resolution of an antibody array is usually directly related to the sheer number of antibodies included and their range of specificities, which tends to be a bottleneck. Third, the numbers of readily available antibodies, designed for microarray applications, have per se been limited. To bypass these technological hurdles and advance further, and even to provide quantitative capabilities, the most attractive features of affinity proteomics and MS could be combined (17, 18). The biological sample would then be digested and exposed to peptide-specific antibodies, after which any specifically enriched peptides would be detected, identified, and potentially quantified using MS. This was exhibited in the stable isotope standard Itgbl1 capture with antipeptide antibodies set-up, but, like conventional antibody arrays, this platform relied on the use of one binder per unique peptide/protein (19, 20). To circumvent the need of having to generate numerous antibodies, we (18) as well as others (17), have recently presented the novel concept of using antibodies directed against short peptide-motifs (epitopes) shared among up to hundreds of different peptides/proteins. This would provide an inherent capability of probing any proteome in a discovery mode, in a species independent manner, while still using a very limited number of antibodies. Based on this concept, we have recently designed a platform denoted global proteome survey (GPS)1, based on human recombinant single-chain fragment variable (scFv) antibodies (18, 22), while Joos presented the triple X proteomic (TXP) set-up, relying on polyclonal and/or monoclonal antibodies. In our case, the scFv antibodies, microarray adapted by molecular design (7, 8, 23), were derived from a large phage-display library (24), representing a renewable probe source displaying an extensive range of specificities. In this manner, one hundred of such scFv antibodies, denoted context-independent motif specific (CIMS) antibodies, could theoretically cover almost 50% of the nonredundant human proteome (18, 22), a concept supported by a recent motif study of the human proteome (25). Recently, we exhibited experimental proof-of-principle for that a limited number of CIMS antibodies could be.