Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome

Background: The oral cavity is home to one of the most diverse microbial communities of the human body and a major entry portal for pathogens. Its homeostasis is maintained by saliva, which fulfills key functions including lubrication of food, pre-digestion, and bacterial defense. Consequently, disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Here we set out to quantitatively map the saliva proteome in great depth with a rapid and in-depth mass spectrometry-based proteomics workflow. Methods: We used recent improvements in mass spectrometry (MS)-based proteomics to develop a rapid workflow for mapping the saliva proteome quantitatively and at great depth. Standard clinical cotton swabs were used to collect saliva form eight healthy individuals at two different time points, allowing us to study interindividual differences and interday changes of the saliva proteome. To accurately identify microbial proteins, we developed a method called "split by taxonomy id" that prevents peptides shared by humans and bacteria or between different bacterial phyla to contribute to protein identification. Results: Microgram protein amounts retrieved from cotton swabs resulted in more than 3700 quantified human proteins in 100-min gradients or 5500 proteins after simple fractionation. Remarkably, our measurements also quantified more than 2000 microbial proteins from 50 bacterial genera. Co-analysis of the proteomics results with next-generation sequencing data from the Human Microbiome Project as well as a comparison to MALDI-TOF mass spectrometry on microbial cultures revealed strong agreement. The oral microbiome differs between individuals and changes drastically upon eating and tooth brushing. Conclusion: Rapid shotgun and robust technology can now simultaneously characterize the human and microbiome contributions to the proteome of a body fluid and is therefore a valuable complement to genomic studies. This opens new frontiers for the study of host-pathogen interactions and clinical saliva diagnostics

Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome

Background: The oral cavity is home to one of the most diverse microbial communities of the human body and a major entry portal for pathogens. Its homeostasis is maintained by saliva, which fulfills key functions including lubrication of food, pre-digestion, and bacterial defense. Consequently, disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Here we set out to quantitatively map the saliva proteome in great depth with a rapid and in-depth mass spectrometry-based proteomics workflow. Methods: We used recent improvements in mass spectrometry (MS)-based proteomics to develop a rapid workflow for mapping the saliva proteome quantitatively and at great depth. Standard clinical cotton swabs were used to collect saliva form eight healthy individuals at two different time points, allowing us to study interindividual differences and interday changes of the saliva proteome. To accurately identify microbial proteins, we developed a method called "split by taxonomy id" that prevents peptides shared by humans and bacteria or between different bacterial phyla to contribute to protein identification. Results: Microgram protein amounts retrieved from cotton swabs resulted in more than 3700 quantified human proteins in 100-min gradients or 5500 proteins after simple fractionation. Remarkably, our measurements also quantified more than 2000 microbial proteins from 50 bacterial genera. Co-analysis of the proteomics results with next-generation sequencing data from the Human Microbiome Project as well as a comparison to MALDI-TOF mass spectrometry on microbial cultures revealed strong agreement. The oral microbiome differs between individuals and changes drastically upon eating and tooth brushing. Conclusion: Rapid shotgun and robust technology can now simultaneously characterize the human and microbiome contributions to the proteome of a body fluid and is therefore a valuable complement to genomic studies. This opens new frontiers for the study of host-pathogen interactions and clinical saliva diagnostics

Exploring skeletal muscle plasticity by single fiber proteomics

Skeletal muscle plasticity involves the transition of muscle fibers through different structural and metabolic phenotypes, to an endpoint matching environmental conditions. The result could be an increase of muscles in mass and performance, as in the case of exercise, or a functional decline, as in the case of age-dependent sarcopenia. We have recently obtained the proteome of single mouse muscle fibers using a liquid chromatography/mass spectrometry-based workflow optimized for low abundant and high dynamic range samples (Murgia et al, EMBO Reports 2015). We here analyse by shotgun proteomics single human muscle fibers from biopsies of healthy subjects differing in age and daily physical activity, as well as of patients with limited mobility. Our results indicate that the proteomes of different fiber types can be clearly distinguished based on structural and metabolic features. Additionally, activity-dependent proteomic features segregate patients\u2019 muscle fibers from those of physically active subjects. Our results will provide important insight into human skeletal muscle plasticity at the level of its cellular units

Parallel Accumulation–Serial Fragmentation (PASEF): Multiplying Sequencing Speed and Sensitivity by Synchronized Scans in a Trapped Ion Mobility Device

In liquid chromatography-mass spectrometry (LC-MS)-based proteomics, many precursors elute from the column simultaneously. In data-dependent analyses, these precursors are fragmented one at a time, whereas the others are discarded entirely. Here we employ trapped ion mobility spectrometry (TIMS) on an orthogonal quadrupole time-of-flight (QTOF) mass spectrometer to remove this limitation. In TIMS, all precursor ions are accumulated in parallel and released sequentially as a function of their ion mobility. Instead of selecting a single precursor mass with the quadrupole mass filter, we here implement synchronized scans in which the quadrupole is mass positioned with sub-millisecond switching times at the <i>m</i>/<i>z</i> values of appropriate precursors, such as those derived from a topN precursor list. We demonstrate serial selection and fragmentation of multiple precursors in single 50 ms TIMS scans. Parallel accumulation–serial fragmentation (PASEF) enables hundreds of MS/MS events per second at full sensitivity. Modeling the effect of such synchronized scans for shotgun proteomics, we estimate that about a 10-fold gain in sequencing speed should be achievable by PASEF without a decrease in sensitivity

Proteomics reveals the effects of sustained weight loss on the human plasma proteome

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill‐defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry‐based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual‐specific protein levels with wide‐ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte‐secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of −16% and +37%, respectively (P < 10(−13)), and the entire apolipoprotein family showed characteristic differential regulation. Clinical laboratory parameters are reflected in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten‐protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly evaluates and monitors intervention in metabolic diseases

Additional file 1: of Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome

Supplementary Figures S1-S9. (PDF 13519 kb

Progressive Multifocal Leukoencephalopathy Treated by Immune Checkpoint Inhibitors

International audienceObjective: Our aim was to assess the real-world effectiveness of immune checkpoint inhibitors for treatment of patients with progressive multifocal leukoencephalopathy (PML).Methods: We conducted a multicenter survey compiling retrospective data from 79 PML patients, including 38 published cases and 41 unpublished cases, who received immune checkpoint inhibitors as add-on to standard of care. One-year follow-up data were analyzed to determine clinical outcomes and safety profile. Logistic regression was used to identify variables associated with 1-year survival.Results: Predisposing conditions included hematological malignancy (n = 38, 48.1%), primary immunodeficiency (n = 14, 17.7%), human immunodeficiency virus/acquired immunodeficiency syndrome (n = 12, 15.2%), inflammatory disease (n = 8, 10.1%), neoplasm (n = 5, 6.3%), and transplantation (n = 2, 2.5%). Pembrolizumab was most commonly used (n = 53, 67.1%). One-year survival was 51.9% (41/79). PML-immune reconstitution inflammatory syndrome (IRIS) was reported in 15 of 79 patients (19%). Pretreatment expression of programmed cell death-1 on circulating T cells did not differ between survivors and nonsurvivors. Development of contrast enhancement on follow-up magnetic resonance imaging at least once during follow-up (OR = 3.16, 95% confidence interval = 1.20-8.72, p = 0.02) was associated with 1-year survival. Cerebrospinal fluid JC polyomavirus DNA load decreased significantly by 1-month follow-up in survivors compared to nonsurvivors (p < 0.0001). Thirty-two adverse events occurred among 24 of 79 patients (30.4%), and led to treatment discontinuation in 7 of 24 patients (29.1%).Interpretation: In this noncontrolled retrospective study of patients with PML who were treated with immune checkpoint inhibitors, mortality remains high. Development of inflammatory features or overt PML-IRIS was commonly observed. This study highlights that use of immune checkpoint inhibitors should be strictly personalized toward characteristics of the individual PML patient. ANN NEUROL 2023;93:257-270