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Citrullination, the Ca2+-driven enzymatic conversion of arginine residues to citrulline, is a posttranslational modification, implicated in several physiological and pathological processes. Several methods to detect citrullinated proteins have been developed, including color development reagent, fluorescence, phenylglyoxal, and antibody-based methods. These methods yet suffer from limitations in sensitivity, specificity, or citrullinated site determination. Mass spectrometry (MS)-based proteomic analysis has emerged as a promising method to resolve these problems. However, due to low abundance of citrullinated proteins and similar MS features to deamidation of asparagine and glutamine, confident identification of citrullinated proteome is challenging. Here, we present a systematic approach to identify a compendium of steps to enhance the number of detected citrullinated residue and implement diagnostic MS feature that allow the confidence of MS-based identifications. Our method is based on the concept of generation of hyper-citrullinated library with high-pH reversed-phase peptide fractionation that allows to enrich in low abundance citrullinated peptides and amplify the effect of charge loss upon citrullination. [https://www.selleckchem.com/products/Everolimus(RAD001).html learn more] Application of our approach to complex global citrullino-proteome datasets demonstrates the confident assessment of citrullinated peptides, thereby enhancing the size and functional interpretation of citrullinated proteomes.The identification of biomarkers for companion diagnostics is revolutionizing the development of treatments tailored to individual patients in different disease areas including cancer. Precision medicine is most frequently based on the detection of genomic markers that correlate with the efficacy of selected targeted therapies. However, since nongenetic mechanisms also contribute to disease biology, there is a considerable interest of using proteomic techniques as additional source of biomarkers to personalize therapies. In this chapter, we describe label-free mass spectrometry methods for proteomic and phosphoproteomic analysis compatible with routine analysis of clinical samples. We also outline bioinformatic pipelines based on statistical learning that use these proteomics datasets as input to quantify kinase activities and predict drug responses in cancer cells.Lysine acetylation is a widespread posttranslational modification (PTM) in all kingdoms of live. A large number of proteins involved in most of biological pathways are targets of this PTM. The lysine acetylation is a reversible modification controlled by two main groups of enzymes, lysine acetyltransferases responsible for transferring the acetyl group of acetylCoA to the side chain of lysine residues and lysine deacetylases which effectively remove the acetyl tag. Dysregulation of enzymes that control acetylation and/or target proteins have been associated with a growing number of human pathologies. Lysine acetylation is largely a modification that occurs at low stoichiometry at its target sites. Here we describe a method to identify lysine acetylation sites and estimate their site occupancy at the proteome scale. The method relies on a high-resolution mass spectrometry-based proteomics approach, which includes a specific chemical acetylation reaction on unmodified lysine residues that carry heavy isotopes. The procedures described here have been applied to cell line cultures and to clinically relevant samples stored as both snap-frozen and formalin-fixed paraffin-embedded (FFPE) tissues.Single-cell proteomics is a novel application area of bioanalysis aiming to characterize proteomes of isolated single cells, which in contrast to bulk cell analysis has the potential to reveal a more detailed heterogeneity of cell populations. Although several antibody-based targeted approaches have been readily available for single-cell analysis, so far only the mass spectrometry methodology can offer unbiased proteome profiling. While this strategy has only recently emerged, it has already demonstrated unparalleled analytical power quantifying &gt;1000 proteins in single cells. Several applications of a general isobaric labeling scheme for multiplexed sample preparation and data acquisition have been outlined using various cell types and instrumentation. This chapter provides a typical example of mass spectrometry-based single-cell proteomics workflow with details about the critical steps of analysis and alternative methods useful for optimization purposes.Human gut microbiota can be studied through the characterization of microorganisms present in feces. Metaproteomics has arisen as a good approach to investigate this vast community. However, the processing of fecal samples in order to obtain the largest number of proteins from gut microbiota to be subsequently analyzed by means of metaproteomics is a challenge. Here we describe a protocol to approach this task. It includes two main steps the first step of humectation and dispersion of the feces, followed by the separation of microorganisms from other fecal components such as roughage and food debris, and the second step in which microbial cells are broken up and microbiota proteins recovered for MS analysis. Detailed procedures for sample preparation, protein extraction, trypsin digestion, and mass spectrometry analysis for gut microbiota samples are provided.Dried blood spots (DBS) are widely used for screening molecular profiles, including enzymatic activity. However, hydrophilic proteins present in large amounts in blood inhibit detection of other proteins in DBS by liquid chromatography-mass spectrometry (LC-MS/MS) without preenrichment. Sodium carbonate precipitation (SCP) can concentrate hydrophobic proteins from DBS and effectively remove soluble hydrophilic proteins. Furthermore, SCP combination with data-independent acquisition (DIA) for quantitative LC-MS/MS enhanced the proteome analysis sensitivity and quantification limits. In this protocol, we have described in detail a simple preenrichment method using SCP and a deep proteome analysis method for LC-MS/MS data using DIA.Mass spectrometry (MS) has become a mainstream platform for comprehensive profiling of proteome, especially with the improvement of multiplexed tandem mass tag labeling coupled with two-dimensional liquid chromatography and tandem mass spectrometry (TMT-LC/LC-MS/MS). Recently, we have established a robust method for direct profiling of undepleted cerebrospinal fluid (CSF) proteome with the 16-plex TMTpro method, in which we optimized parameters in experimental steps of sample preparation, TMT labeling, LC/LC fractionation, tandem mass spectrometry, and computational data processing. The extensive LC fractionation not only enhances proteome coverage of the CSF but also alleviates ratio distortion of TMT quantification. The crucial quality control steps and improvements specific for the TMT16 analysis are highlighted. More than 3000 proteins can be quantified in a single experiment from 16 different CSF samples. This multiplexed method offers a powerful tool for profiling a variety of complex biofluids samples such as CSF, serum/plasma, and other clinical specimens.Parasite infections caused by helminths affect hundreds of millions worldwide. Despite their socioeconomic importance and impact on health, there is still an urgent need to develop appropriate control approaches. The recent discovery that helminths, as most eukaryotic organisms, secrete extracellular vesicles (EVs) of different type has opened new avenues for the characterization of novel diagnostic and vaccine candidates that could serve for this purpose. Herein, we describe a method for the isolation of highly pure microvesicles and exosomes, two of the most relevant populations of EVs secreted by helminths, and describe a validated approach to characterize the proteins from different compartments of EVs. These proteins could be further developed into suitable diagnostic and vaccine candidates against these devastating infections.One of the critical issues to warrant the success of a proteome-wide analysis is sample preparation. Efficient protein extraction in the absence of interferent material is mandatory to achieve an ample proteome coverage by mass spectrometry. The study of biological fluids is always challenging due to their specific biochemical composition. However, there is increasing interest in their characterization as it will provide proteins that may advice disease setting, state, and progression. In particular, bile is proximal to liver and pancreas, and its study is especially attractive since it might provide valuable information for the clinical management of severe diseases afflicting these organs, which are at an urgent need of new biomarkers. Though previous efforts have been made to optimize protocols to analyze bile proteome, only partial descriptions were achieved due to its complex composition, where proteins represent less than 5% of the organic components. Here we describe a new method that significantly increases the bile proteome coverage while reducing by a factor of six the amount of sample required for the proteomic analysis.<br /><br /> Chorea, a common clinical manifestation of Huntington's disease (HD), involves sudden, involuntary movements that interfere with daily functioning and contribute to the morbidity of HD. Tetrabenazine and deutetrabenazine are FDA-approved to treat chorea associated with HD. Compared to tetrabenazine, deutetrabenazine has a unique pharmacokinetic profile leading to more consistent systemic exposure, less frequent dosing, and a potentially more favorable safety/tolerability profile. Real-world adherence data for these medications are limited. Here, we evaluate real-world adherence patterns with the vesicular monoamine transporter 2 inhibitors, tetrabenazine and deutetrabenazine, among patients diagnosed with HD.<br /><br /> Insurance claims data from the Symphony Health Solutions Integrated Dataverse (05/2017-05/2019) were retrospectively analyzed for patients diagnosed with HD (ICD-10-CM code G10). Patients were categorized into cohorts based on treatment. Outcomes included adherence, which was measured by proportion oe tetrabenazine cohort. However, a potential limitation is overestimated adherence, as claims for prescription fills may not capture actual use. Additional research is warranted to explore the differences in adherence patterns between treatments, which may inform treatment decision-making.<br /><br />Results from this real-world analysis indicate that patients treated with deutetrabenazine are more adherent to treatment and have lower discontinuation rates compared with patients in the tetrabenazine cohort. However, a potential limitation is overestimated adherence, as claims for prescription fills may not capture actual use. Additional research is warranted to explore the differences in adherence patterns between treatments, which may inform treatment decision-making.<br /><br /> This study investigated the long-term visual and optical quality changes after small incision lenticule extraction (SMILE) for the correction of moderate to high myopia.<br /><br /> Forty-one eyes of 41 patients with a mean spherical equivalent (SE) of -6.74 ± 1.45 D undergoing SMILE were enrolled. Preoperative and 1-month, 3-month, 1-year and 5-year postoperative follow-up examinations were performed including visual acuity, manifest refraction, corneal aberrations and contrast sensitivity. An optical quality analysis system was used to measure objective scatter index (OSI), Strehl ratio (SR) and modulation transfer function cutoff frequency (MTF<br /><br /> ).<br /><br /> The safety and efficacy indices were 1.09 ± 0.12 and 1.03 ± 0.17, respectively, at the 5-year follow-up. No significant changes in SE from 1month to 5years postoperatively were found. The OSI significantly increased at 1 and 3months postoperatively (p &lt; 0.01 and p &lt; 0.01) and declined at 1 and 5years (p = 0.81 and p = 0.87). MTF<br /><br /> and SR showed similar trends. Coma, spherical aberration and total higher-order aberration significantly increased after SMILE (p &lt; 0.