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Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g.candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches.

Using global liquid chromatography-mass spectrometry (LC-MS)–based proteomics analyses, we identified 24 serum proteins that were significantly variant between those with type 1 diabetes (T1D) and healthy controls. Functionally, these proteins represent innate immune responses, the activation cascade of complement, inflammatory responses, and blood coagulation. Targeted verification analyses were performed on 52 surrogate peptides representing these proteins, with serum samples from an antibody standardization program cohort of 100 healthy control and 50 type 1 diabetic subjects. 16 peptides were verified as having very good discriminating power, with areas under the receiver operating characteristic curve ≥0.8. Further validation with blinded serum samples from an independent cohort (10 healthy control and 10 type 1 diabetics) demonstrated that peptides from platelet basic protein and C1 inhibitor achieved both 100% sensitivity and 100% specificity for classification of samples. The disease specificity of these proteins was assessed using sera from 50 age-matched type 2 diabetic individuals, and a subset of proteins, C1 inhibitor in particular, were exceptionally good discriminators between these two forms of diabetes. The panel of biomarkers distinguishing those with T1D from healthy controls and those with type 2 diabetes suggests that dysregulated innate immune responses may be associated with the development of this disorder.

Salmonella entericaserovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes ∼8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. entericagenome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based “bottom-up” proteomic approach to generate a more complete picture of the gene products that S. typhimuriumsynthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimuriumis known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimuriumexpress genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg2+transport proteins than found in other growth conditions. A second more virulent S. typhimuriumstrain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pduoperon, which encodes enzymes required for propanediol utilization. These pduoperon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdugene expression contributes to S. typhimuriumpathogenesis.