Project description:This study investigates the efficacy of proteomic analysis of human remains to identify active Mycobacterium leprae infections in the past. Mycobacterial diseases, like leprosy, have plagued human populations for millennia. Thanks to effective treatment options, leprosy is not as widespread and deadly as in the past, yet remains endemic in certain regions with increasing concerns of strains becoming resistant to antibiotic treatments. We present a dual-enzyme, optimised extraction protocol, using trypsin and ProAlanase, to increase the recovery of non-collagenous proteins through a study of five individuals from a Mediaeval leprosarium cemetery, as well as four from a non-leprosy associated cemetery. Here we show that skeletal samples from the leprosarium individuals contain numerous immune proteins associated with modern leprosy, while those from a non-leprosy associated cemetery do not. Through this study, we advance a palaeoimmunology methodology and provide insights into the health of archaeological individuals and offer a means to triage samples for aDNA analysis.
Project description:The enamel proteome includes a range of proteins which are well-preserved in archaeological settings but have so far received less study than the sex-specific sequencing of enamel. We look beyond sex-specific sequencing of amelogenin to investigate the potential of several serum proteins, including immunoglobulin gamma (IgG), the major immunoglobulin found in blood serum, and C-reactive protein (CRP) which is associated with inflammatory response, to provide insight into the health and stresses experienced by individuals in the past. We apply this approach to enamel samples from Mission-Period ancestral Ohlone interred at Asistencia San Pedro y San Pablo (CA-SMA-71/H; n=11). For comparison, we also examine enamel from historic-period European-Americans interred in the City Cemetery in San Francisco, and extracted third molars from present-day military cadets. Results indicate that IgG is elevated among individuals at the asistencia relative to samples from present-day military cadets (n=8), or historic City Cemetery individuals (ANOVA with post-hoc Tukey Kramer tests, p < 0.02). Further, the inflammatory protein CRP, normally expressed at much lower levels than IgG, was present in 55% (6 of 11) of the asistencia samples, and in 17% (2 of 12) of the historic City Cemetery samples, but was not detected in enamel samples from contemporary military cadets. While more studies are needed, we argue that the difference in IgG could reflect higher levels of chronic diseases such as tuberculosis among Ohlone living within the Mission system, while the presence of measurable amounts of CRP could relate to high degrees of physical, social, and emotional stresses. To our knowledge, this is the first paleoproteomic study of immune proteins in tooth enamel. The ability to track immune responses during tooth formation could provide valuable and high-resolution information on ancient health and disease at the level of the individual over archaeological time-scales.
Project description:8 leprosy patients including 4 multibacillary (MB) and 4 paucibacillary (PB), and 8 non-leprosy controls including 4 healthy house contacts (HHCs) and 4 endemic controls (ECs) were included in the study. The immune response differences between leprosy patients and controls were evaluated by analyzing the transcriptional profiles of PBMCs to M. leprae sonicate antigens by RNA-seq. The analyses revealed potential biomarkers (including mRNAs and lncRNAs) preferentially expressed in PBMCs in leprosy patients that may be useful for early diagnosis of leprosy.
Project description:Multiplexing approaches using tandem mass tags with a carrier proteome to boost sensitivity have advanced single cell proteomics by mass spectrometry (SCoPE-MS). Here, we probe the carrier proteome effects in single cell proteomics with mixed species TMTpro-labeled samples. We demonstrate that carrier proteomes, while increasing overall identifications, dictate which proteins are identified. We show that quantitative precision and signal intensity are limited at high carrier levels, hindering the recognition of regulated proteins. Guidelines for optimized mass spectrometry acquisition parameters and best practices for fold-change or protein copy number-based comparisons are provided.
Project description:Transcriptomics and proteomics analyses were carried out on rectal mucosal biopsies from 22 healthy adults with either optimal (n=11, mean plasma Se =1.43μmol/l, s.e.m ±0.06) or sub-optimal (n=11, mean plasma Se = 0.86umol/l, s.e.m ± 0.01) plasma Se status. Plasma Se status was associated with altered expression of 254 genes implicated in cancer, immune function and inflammatory response, cell growth and proliferation, cellular movement and cell death; 69 out of 254 genes had their expression significantly correlated with Se status, including two selenoprotein genes (SEPW1 and SELK). Proteomics identified 26 proteins differentially expressed between the two Se groups, including cytoskeletal proteins and factors involved in immune and inflammatory response and cancer pathways. Results from proteomics and transcriptomics support each other and integration of the two datasets was consistent with a reduced inflammatory and immune responses and a cytoskeleton remodelling in the rectal mucosa of individuals with sub-optimal Se status.
Project description:Inflammaging is the name given to this chronic and asymptomatic inflammatory state generated by the aging process and by chronic and infectious diseases. Chronic diseases can alter the epigenetic profile of tissues leading to an increased epigenetic aging and some of the differentially methylated genes can be used to characterize the disease and as disease biomarkers. Leprosy is an infection caused by Mycobacterium leprae and can be lifelong, exposing the individual to a low-grade inflammation environment. In this study, we evaluated the inflammatory profile in 35 individuals from a leprosy endemic area from Brazil by cytokine analysis with a luminex assay. In adition, we investigated the leucocytes genome-wide DNA methylation profile using the Infinium MethylationEPIC BeadChip array. A total number of 31 CpGs were significantly methylated, between cases and controls, which belonged to 8 genes potentially peripherally perturbed in the pathogenesis of the disease. Affected individuals from endemic area were epigenetically aged in relation to control samples and interesting control samples from endemic area were aged in relation to unaffected control samples from non-endemic area. In conclusion, leprosy showed a deregulated methylation profile in comparison with control samples. The epigenetic analysis provided valuable clues for further investigations in understanding peripherical blood leprosy alterations and the use of these genes as biomarkers.
Project description:Field asymmetric ion mobility spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity and enables more proteins to be identified by providing additional gas-phase separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications, an 8% increase in proteoform identifications, and an improvement in proteoform identification compared to samples analyzed without FAIMS. In addition, utilization of FAIMS resulted in the identification of proteins encoded by lower-abundance mRNA transcripts. These improvements were attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications.
Project description:FAMIN (LACC1, C13orf31) loss-of-function causes systemic juvenile idiopathic arthritis and very early-onset inflammatory bowel disease, while a common I254V substitution results in hypomorphic function and increases susceptibility to Crohn’s disease and leprosy. In this study, we compare the mRNA transcriptional profiles of M0, M1 and M2-polarised bone marrow-derived macrophages from mice engineered at their endogenous locus to express human non-risk (Faminp.254I), risk (Faminp.254V) and monogenic (Faminp.284R) disease variants.
Project description:Data-dependent precursor ion selection is widely used in shotgun proteomics to profile the protein components of complex samples. Although very popular, this bottom-up method presents major drawbacks in terms of detectable dynamic range. Here, we demonstrate the superior performance of a data-independent method we term precursor acquisition independent from ion count (PAcIFIC). Our results show that almost the entire, predicted, soluble bacterial proteome can be thoroughly analyzed by PAcIFIC without the need for any sample fractionation other than the C18-based liquid chromatograph used to introduce the peptide mixture into the mass spectrometer. Importantly, we also show that PAcIFIC provides unique performance for analysis of human plasma in terms of the number of proteins identified (746 at FDR < or = 0.5%) and achieved dynamic range (8 orders of magnitude at FDR < or = 0.5%), without any fractionation other than immuno-depletion of the seven most abundant proteins.