Oct-B: A derivative of L-BAIBA significantly alleviating high-fat diet-induced obesity in mice.

The rising prevalence of obesity is a global health concern. Supplementation with (S)-ß-aminoisobutyric acid (L-BAIBA) has shown potential in preventing obesity and related metabolic disorders induced by high-fat diets. However, developing effective and low-toxicity BAIBA derivatives remains a challenging yet promising field. In this study, we introduce Oct-B, a novel BAIBA ester compound, which exhibits 80-fold greater efficacy than L-BAIBA in alleviating obesity in high-fat diet-fed mice. Our results demonstrate that Oct-B significantly reduces serum TG, TC, LDL-C, and the activities of ALT and AST, and also reduces TG and TC in liver, surpassing the effects of L-BAIBA. Histological analysis shows that Oct-B significantly decreases lipid accumulation in liver tissues, normalizes mast cells in white adipose tissue, and upregulates the expression of UCP1 protein in white adipose tissue. The qRT-PCR results indicated Oct-B alleviates obesity by downregulating lipogenic genes (PPARγ, ACC1, FAS), upregulating lipolysis related genes (PPARα, HSL) and thermogenic gene UCP1. Additionally, quantitative mass spectrometry reveals a marked increase in L-BAIBA levels in white fat, brown fat, serum, and muscle following Oct-B administration. These findings suggest that Oct-B is an efficient L-BAIBA substitute, offering a promising therapeutic approach for preventing and treating high-fat diet-induced obesity.

Novel mimetic tissue standards for precise quantitative mass spectrometry imaging of drug and neurotransmitter concentrations in rat brain tissues.

Understanding the relationship between the concentration of a drug and its therapeutic efficacy or side effects is crucial in drug development, especially to understand therapeutic efficacy in central nervous system drug, quantifying drug-induced site-specific changes in the levels of endogenous metabolites, such as neurotransmitters. In recent times, evaluation of quantitative distribution of drugs and endogenous metabolites using matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) has attracted much attention in drug discovery research. However, MALDI-MSI quantification (quantitative mass spectrometry imaging, QMSI) is an emerging technique, and needs to be further developed for practicable and convenient use in drug discovery research. In this study, we developed a reliable QMSI method for quantification of clozapine (antipsychotic drug) and dopamine and its metabolites in the rat brain using MALDI-MSI. An improved mimetic tissue model using powdered frozen tissue for QMSI was established as an alternative method, enabling the accurate quantification of clozapine levels in the rat brain. Furthermore, we used the improved method to evaluate drug-induced fluctuations in the concentrations of dopamine and its metabolites. This method can quantitatively evaluate drug localization in the brain and drug-induced changes in the concentration of endogenous metabolites, demonstrating the usefulness of QMSI.

Quantitative mass spectrometry analysis of the injured proximal and distal human digital nerve ends.

Introduction: Proteomic analysis of injured human peripheral nerves, particularly focusing on events occurring in the proximal and distal nerve ends, remains relatively underexplored. This study aimed to investigate the molecular patterns underlying a digital nerve injury, focusing on differences in protein expression between the proximal and distal nerve ends. Methods: A total of 26 human injured digital nerve samples (24 men; 2 women; median age 47 [30-66] years), harvested during primary nerve repair within 48 h post-injury from proximal and distal nerve ends, were analyzed using mass spectrometry. Results: A total of 3,914 proteins were identified, with 127 proteins showing significant differences in abundance between the proximal and the distal nerve ends. The downregulation of proteins in the distal nerve end was associated with synaptic transmission, autophagy, neurotransmitter regulation, cell adhesion and migration. Conversely, proteins upregulated in the distal nerve end were implicated in cellular stress response, neuromuscular junction stability and muscle contraction, neuronal excitability and neurotransmitter release, synaptic vesicle recycling and axon guidance and angiogenesis. Discussion: Investigation of proteins, with functional annotations analysis, in proximal and the distal ends of human injured digital nerves, revealed dynamic cellular responses aimed at promoting tissue degeneration and restoration, while suppressing non-essential processes.

Proteostasis as a fundamental principle of Tau immunotherapy.

The microtubule-associated protein Tau is a driver of neuronal dysfunction in Alzheimer's disease and other tauopathies. In this process, Tau initially undergoes subtle changes to its abundance, subcellular localisation and a vast array of post-translational modifications including phosphorylation, that progressively result in the protein's somatodendritic accumulation and dysregulation of multiple Tau-dependent cellular processes. Given the various loss- and gain-of-functions of Tau in disease and the brain-wide changes in the proteome that characterise tauopathies, we asked whether targeting Tau would restore the alterations in proteostasis observed in disease. Therefore, by phage display, we generated a novel pan-Tau antibody, RNJ1, that preferentially binds human Tau and neutralises proteopathic seeding activity in multiple cell lines, and benchmarked it against a clinically tested pan-Tau antibody, HJ8.5 (murine version of tilavonemab). We then evaluated both antibodies, alone and in combination, in the K3 tauopathy mouse model, showing reduced Tau pathology and improvements in neuronal function following 14 weekly treatments, without obtaining synergy for the combination. These effects were more pronounced in female mice. To investigate the molecular mechanisms contributing to improvements in neuronal function, we employed quantitative proteomics, phosphoproteomics and kinase prediction analysis to first establish alterations in K3 mice relative to WT controls at the proteome level. In female K3 mice, we found 342 differentially abundant proteins, which are predominantly involved in metabolic and microtubule-associated processes, strengthening previously reported findings of defects in several functional domains in multiple tauopathy models. We next asked whether antibody-mediated Tau target engagement indirectly affects levels of deregulated proteins in the K3 model. Importantly, both immunotherapies, in particular RNJ1, induced abundance shifts towards a restoration to wild-type levels (proteostasis). A total of 257 of 342 (∼75%) proteins altered in K3 were closer in abundance to WT levels after RNJ1 treatment, and 73% after HJ8.5 treatment. However, the magnitude of these changes was less pronounced than that observed with RNJ1, as reflected by a far smaller number of differentially abundant proteins. Furthermore, analysis of the phosphoproteome showed an even stronger restoration effect with RNJ1, with ∼82% of altered phosphopeptides in K3 showing a shift to WT levels, and 75% with HJ8.5. Gene set over-representation analysis (ORA) further confirmed that proteins undergoing restoration are involved in biological pathways affected in K3 mice. Together, our study suggests that a Tau immunotherapy-induced restoration of proteostasis links target engagement and treatment efficacy.

Extraction of Proteins from Soil.

Soil metaproteomics could explore the proteins involved in life activities and their abundance in the soils to overcome the difficulty in pure cultures of soil microorganisms and the limitations of proteomics of pure cultures. However, the complexity and heterogeneity of soil composition, the low abundance of soil proteins, and the presence of massive interfering substances (including humic compounds) generally lead to an extremely low extraction efficiency of soil proteins. Therefore, the efficient extraction of soil proteins is a prerequisite and bottleneck problem in soil metaproteomics. In this chapter, a soil protein extraction method suitable for most types of soils with low cost and enabling simple operation (about 150 µg protein can be extracted from 5.0 g soil) is described. The quantity and purity of the extracted soil proteins could meet the requirements for further analysis using routine mass spectrometry-based proteomics.

Inserting Pre-analytical Chromatographic Priming Runs Significantly Improves Targeted Pathway Proteomics with Sample Multiplexing.

GoDig, a platform for targeted pathway proteomics without the need for manual assay scheduling or synthetic standards, is a powerful, flexible, and easy-to-use method that uses tandem mass tags to increase sample throughput up to 18-fold relative to label-free methods. Though the protein-level success rates of GoDig are high, the peptide-level success rates are more limited, hampering assays of harder-to-quantify proteins and site-specific phenomena. To guide the optimization of GoDig assays as well as improvements to the GoDig platform, we created GoDigViewer, a new stand-alone software that provides detailed visualizations of GoDig runs. GoDigViewer guided the implementation of "priming runs," an acquisition mode with significantly higher success rates. In this mode, two or more chromatographic priming runs are automatically performed to improve the accuracy and precision of target elution orders, followed by analytical runs which quantify targets. Using priming runs, success rates exceeded 97% for a list of 400 peptide targets and 95% for a list of 200 targets that are usually not quantified using untargeted mass spectrometry. We used priming runs to establish a quantitative assay of 125 macroautophagy proteins that had a >95% success rate and revealed differences in macroautophagy expression profiles across four human cell lines.

Motilin fluctuations in healthy volunteers determined by liquid chromatography mass spectrometry.

Introduction: Motilin is a hormone secreted by specialised enteroendocrine cells in the small intestine, and is known to modulate gastrointestinal motility in humans, regulating the migratory motor complex. It is understudied at least in part due to the lack of commercially available immunoassays. Method: A multiplexed liquid chromatography mass spectrometry (LC-MS/MS) method was optimised to measure motilin, insulin, C-peptide, GIP (1-42) and GIP (3-42). Corresponding active ghrelin concentrations were determined by immunoassay. Ten healthy volunteers with no prior history of gastroenterological or endocrine condition attended after overnight fast and had blood samples taken every 15 minutes for 4 hours whilst continuing to fast, and then further sampling for 2 hours following a liquid mixed meal. Hunger scores were taken at each time point using a visual analogue scale. Normal bowel habit was confirmed by 1 week stool diary. Results: Motilin levels fluctuated in the fasting state with an average period between peaks of 109.5 mins (SD:30.0), but with no evidence of a relationship with either ghrelin levels or hunger scores. The mixed meal interrupted cyclical motilin fluctuations, increased concentrations of motilin, insulin, C-peptide, GIP(1-42) and GIP(3-42), and suppressed ghrelin levels. Discussion: This study highlights the utility of LC-MS/MS for parallel measurement of motilin alongside other peptide hormones, and supports previous reports of the cyclical nature of motilin levels in the fasting state and interruption with feeding. This analytical method has utility for further clinical studies into motilin and gut hormone physiology in human volunteers.

The Proteomic Composition and Organization of Constitutive Heterochromatin in Mouse Tissues.

Pericentric heterochromatin (PCH) forms spatio-temporarily distinct compartments and affects chromosome organization and stability. Albeit some of its components are known, an elucidation of its proteome and how it differs between tissues in vivo is lacking. Here, we find that PCH compartments are dynamically organized in a tissue-specific manner, possibly reflecting compositional differences. As the mouse brain and liver exhibit very different PCH architecture, we isolated native PCH fractions from these tissues, analyzed their protein compositions using quantitative mass spectrometry, and compared them to identify common and tissue-specific PCH proteins. In addition to heterochromatin-enriched proteins, the PCH proteome includes RNA/transcription and membrane-related proteins, which showed lower abundance than PCH-enriched proteins. Thus, we applied a cut-off of PCH-unspecific candidates based on their abundance and validated PCH-enriched proteins. Amongst the hits, MeCP2 was classified into brain PCH-enriched proteins, while linker histone H1 was not. We found that H1 and MeCP2 compete to bind to PCH and regulate PCH organization in opposite ways. Altogether, our workflow of unbiased PCH isolation, quantitative mass spectrometry, and validation-based analysis allowed the identification of proteins that are common and tissue-specifically enriched at PCH. Further investigation of selected hits revealed their opposing role in heterochromatin higher-order architecture in vivo.

Differential superoxide production in phosphorylated neuronal nitric oxide synthase mu and alpha variants.

Neuronal nitric oxide synthase (nNOS) is regulated by phosphorylation in vivo, yet the underlying biochemical mechanisms remain unclear, primarily due to difficulty in obtaining milligram quantities of phosphorylated nNOS protein; detailed spectroscopic and rapid kinetics investigations require purified protein samples at a concentration in the range of hundreds microM. Moreover, the functional diversity of the nNOS isoform is linked to its splice variants. Also of note is that determination of protein phosphorylation stoichiometry remains as a challenge. To address these issues, this study first expanded a recent genetic code expansion approach to produce phosphorylated rat nNOSµ and nNOSα holoproteins through site-specific incorporation of phosphoserine (pSer) at residues 1446 and 1412, respectively; this site is at the C-terminal tail region, a NOS-unique regulatory element. A quantitative mass spectrometric approach was then developed in-house to analyze unphosphorylated peptides in phosphatase-treated and -untreated phospho-nNOS proteins. The observed pSer-incorporation efficiency consistently exceeded 80%, showing high pSer-incorporation efficiency. Notably, EPR spin trapping results demonstrate that under l-arginine-depleted conditions, pSer1412 nNOSα presented a significant reduction in superoxide generation, whereas pSer1446 nNOSµ exhibited the opposite effect, compared to their unphosphorylated counterparts. This suggests that phosphorylation at the C-terminal tail has a regulatory effect on nNOS uncoupling that may differ between variant forms. Furthermore, the methodologies for incorporating pSer into large, complex protein and quantifying the percentage of phosphorylation in recombinant purified protein should be applicable to other protein systems.

Evaluating Proteomics Imputation Methods with Improved Criteria.

Quantitative measurements produced by tandem mass spectrometry proteomics experiments typically contain a large proportion of missing values. Missing values hinder reproducibility, reduce statistical power, and make it difficult to compare across samples or experiments. Although many methods exist for imputing missing values, in practice, the most commonly used methods are among the worst performing. Furthermore, previous benchmarking studies have focused on relatively simple measurements of error such as the mean-squared error between imputed and held-out values. Here we evaluate the performance of commonly used imputation methods using three practical, "downstream-centric" criteria. These criteria measure the ability to identify differentially expressed peptides, generate new quantitative peptides, and improve the peptide lower limit of quantification. Our evaluation comprises several experiment types and acquisition strategies, including data-dependent and data-independent acquisition. We find that imputation does not necessarily improve the ability to identify differentially expressed peptides but that it can identify new quantitative peptides and improve the peptide lower limit of quantification. We find that MissForest is generally the best performing method per our downstream-centric criteria. We also argue that existing imputation methods do not properly account for the variance of peptide quantifications and highlight the need for methods that do.

Involvement of the AKT Pathway in Resistance to Erlotinib and Cabozantinib in Triple-Negative Breast Cancer Cell Lines.

Resistance to protein tyrosine kinase inhibitors (TKIs) presents a significant challenge in therapeutic target development for cancers such as triple-negative breast cancer (TNBC), where conventional therapies are ineffective at combatting systemic disease. Due to increased expression, the receptor tyrosine kinases EGFR (epidermal growth factor receptor) and c-Met are potential targets for treatment. However, targeted anti-EGFR and anti-c-Met therapies have faced mixed results in clinical trials due to acquired resistance. We hypothesize that adaptive responses in regulatory kinase networks within the EGFR and c-Met signaling axes contribute to the development of acquired erlotinib and cabozantinib resistance. To test this, we developed two separate models for cabozantinib and erlotinib resistance using the MDA-MB-231 and MDA-MB-468 cell lines, respectively. We observed that erlotinib- or cabozantinib-resistant cell lines demonstrate enhanced cell proliferation, migration, invasion, and activation of EGFR or c-Met downstream signaling (respectively). Using a SILAC (Stable Isotope Labeling of Amino acids in Cell Culture)-labeled quantitative mass spectrometry proteomics approach, we assessed the effects of erlotinib or cabozantinib resistance on the phosphoproteome, proteome, and kinome. Using this integrated proteomics approach, we identified several potential kinase mediators of cabozantinib resistance and confirmed the contribution of AKT1 to erlotinib resistance in TNBC-resistant cell lines.

Reverse-ChIP Techniques for Identifying Locus-Specific Proteomes: A Key Tool in Unlocking the Cancer Regulome.

A phenotypic hallmark of cancer is aberrant transcriptional regulation. Transcriptional regulation is controlled by a complicated array of molecular factors, including the presence of transcription factors, the deposition of histone post-translational modifications, and long-range DNA interactions. Determining the molecular identity and function of these various factors is necessary to understand specific aspects of cancer biology and reveal potential therapeutic targets. Regulation of the genome by specific factors is typically studied using chromatin immunoprecipitation followed by sequencing (ChIP-Seq) that identifies genome-wide binding interactions through the use of factor-specific antibodies. A long-standing goal in many laboratories has been the development of a 'reverse-ChIP' approach to identify unknown binding partners at loci of interest. A variety of strategies have been employed to enable the selective biochemical purification of sequence-defined chromatin regions, including single-copy loci, and the subsequent analytical detection of associated proteins. This review covers mass spectrometry techniques that enable quantitative proteomics before providing a survey of approaches toward the development of strategies for the purification of sequence-specific chromatin as a 'reverse-ChIP' technique. A fully realized reverse-ChIP technique holds great potential for identifying cancer-specific targets and the development of personalized therapeutic regimens.

The interplay of post-translational protein modifications in Arabidopsis leaves during photosynthesis induction.

Diurnal dark to light transition causes profound physiological changes in plant metabolism. These changes require distinct modes of regulation as a unique feature of photosynthetic lifestyle. The activities of several key metabolic enzymes are regulated by light-dependent post-translational modifications (PTM) and have been studied at depth at the level of individual proteins. In contrast, a global picture of the light-dependent PTMome dynamics is lacking, leaving the response of a large proportion of cellular function undefined. Here, we investigated the light-dependent metabolome and proteome changes in Arabidopsis rosettes in a time resolved manner to dissect their kinetic interplay, focusing on phosphorylation, lysine acetylation, and cysteine-based redox switches. Of over 24 000 PTM sites that were detected, more than 1700 were changed during the transition from dark to light. While the first changes, as measured 5 min after onset of illumination, occurred mainly in the chloroplasts, PTM changes at proteins in other compartments coincided with the full activation of the Calvin-Benson cycle and the synthesis of sugars at later timepoints. Our data reveal connections between metabolism and PTM-based regulation throughout the cell. The comprehensive multiome profiling analysis provides unique insight into the extent by which photosynthesis reprograms global cell function and adds a powerful resource for the dissection of diverse cellular processes in the context of photosynthetic function.

Comparing 13C methyl and deuterated methyl isotopic labeling for the quantification of methyl cellulose patterns using mass spectrometry.

The methyl substitution along and among the polymer chains of methyl cellulose (MC) is commonly analyzed by ESI-MS after perdeuteromethylation of the free-OH groups and partial hydrolysis to cello-oligosaccharides (COS). This method requires a correct quantification of the molar ratios of the constituents belonging to a particular degree of polymerization (DP). However, isotopic effects are most pronounced for H/D since their mass difference is 100%. Therefore, we investigated whether more precise and accurate results could be obtained for the methyl distribution of MC by MS of 13CH3 instead of CD3-etherified O-Me-COS. Internal isotope labeling with 13CH3 makes the COS of each DP chemically and physically much more similar, reducing mass fractionation effects, but at the same time requires more complex isotopic correction for evaluation. Results from syringe pump infusion ESI-TOF-MS with 13CH3 and CD3 as isotope label were equal. However, in the case of LC-MS with a gradient system, 13CH3 was superior to CD3. In the case of CD3, the occurrence of a partial separation of the isotopologs of a particular DP resulted in slight distortion of the methyl distribution since the signal response is significantly dependent on the solvent composition. Isocratic LC levels this problem, but one particular eluent-composition is not sufficient for a series of oligosaccharides with increasing DP due to peak broadening. In summary, 13CH3 is more robust to determine the methyl distribution of MCs. Both syringe pump and gradient-LC-MS measurements are possible, and the more complex isotope correction is not a disadvantage.

Label-free quantitative mass spectrometry analysis of the circadian proteome of Drosophila melanogaster lethal giant larvae mutants reveals potential therapeutic effects of melatonin.

Mutation in the Drosophila melanogaster lethal giant larvae (lgl), a tumor suppressor gene with a well-established role in cellular polarity, is known to results in massive cellular proliferation and neoplastic outgrowths. Although the tumorigenic properties of lgl mutant have been previously studied, however, little is known about its consequences on the proteome. In this study, mass spectrometry-based label-free quantitative proteomics was employed to investigate the changes in the head and intestinal tissues proteins of Drosophila melanogaster, due to lgl mutation and following treatment with melatonin. Additionally, to uncover the time-influenced variations in the proteome during tumorigenesis and melatonin treatment, the rhythmic expression of proteins was also investigated at 6-h intervals within 24-h clock. Together, the present study has identified 434 proteins of altered expressions (p < 0.05 and fold change ±1.5) in the tissues of flies in response to lgl mutation as well as posttreatment with melatonin. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed proteins revealed that lgl mutation had significantly affected the biological functions, including metabolism, and protein synthesis and degradation, in flies' tissues. Besides, melatonin had beneficially mitigated the deleterious effects of lgl mutation by reversing the alterations in protein expression closer to baseline levels. Further, changes in protein expression in the tissues due to lgl mutation and melatonin treatment were found rhythmically orchestrated. Together, these findings provide novel insight into the pathways involved in lgl-induced tumorigenesis as well as demonstrated the efficacy of melatonin as a potential anticancer agent. Data are available via ProteomeXchange with identifier PXD033191.

Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells.

In endothelial cells (ECs), stimulation of Toll-like receptor 4 (TLR4) by the endotoxin lipopolysaccharide (LPS) induces the release of diverse pro-inflammatory mediators, beneficial in controlling bacterial infections. However, their systemic secretion is a main driver of sepsis and chronic inflammatory diseases. Since distinct and rapid induction of TLR4 signaling is difficult to achieve with LPS due to the specific and non-specific affinity to other surface molecules and receptors, we engineered new light-oxygen-voltage-sensing (LOV)-domain-based optogenetic endothelial cell lines (opto-TLR4-LOV LECs and opto-TLR4-LOV HUVECs) that allow fast, precise temporal, and reversible activation of TLR4 signaling pathways. Using quantitative mass-spectrometry, RT-qPCR, and Western blot analysis, we show that pro-inflammatory proteins were not only expressed differently, but also had a different time course when the cells were stimulated with light or LPS. Additional functional assays demonstrated that light induction promoted chemotaxis of THP-1 cells, disruption of the EC monolayer and transmigration. In contrast, ECs incorporating a truncated version of the TLR4 extracellular domain (opto-TLR4 ΔECD2-LOV LECs) revealed high basal activity with fast depletion of the cell signaling system upon illumination. We conclude that the established optogenetic cell lines are well suited to induce rapid and precise photoactivation of TLR4, allowing receptor-specific studies.

Proteome-wide lysine acetylation profiling to investigate the involvement of histone deacetylase HDA5 in the salt stress response of Arabidopsis leaves.

Post-translational modifications (PTMs) of proteins play important roles in the acclimation of plants to environmental stress. Lysine acetylation is a dynamic and reversible PTM, which can be removed by histone deacetylases. Here we investigated the role of lysine acetylation in the response of Arabidopsis leaves to 1 week of salt stress. A quantitative mass spectrometry analysis revealed an increase in lysine acetylation of several proteins from cytosol and plastids, which was accompanied by altered histone deacetylase activities in the salt-treated leaves. While activities of HDA14 and HDA15 were decreased upon salt stress, HDA5 showed a mild and HDA19 a strong increase in activity. Since HDA5 is a cytosolic-nuclear enzyme from the class II histone deacetylase family with yet unknown protein substrates, we performed a lysine acetylome analysis on hda5 mutants and characterized its substrate proteins. Next to histone H2B, the salt stress-responsive transcription factor GT2L and the dehydration-related protein ERD7 were identified as HDA5 substrates. In addition, in protein-protein interaction studies, HDA18 was discovered, among other interacting proteins, to work in a complex together with HDA5. Altogether, this study revealed the substrate proteins of HDA5 and identified new lysine acetylation sites which are hyperacetylated upon salt stress. The identification of specific histone deacetylase substrate proteins, apart from histones, will be important to unravel the acclimation response of Arabidopsis to salt stress and their role in plant physiology.

Quantitative structural proteomics in living cells by covalent protein painting.

The fold and conformation of proteins are key to successful cellular function, but all techniques for protein structure determination are performed in an artificial environment with highly purified proteins. While protein conformations have been solved to atomic resolution and modern protein structure prediction tools rapidly generate near accurate models of proteins, there is an unmet need to uncover the conformations of proteins in living cells. Here, we describe Covalent Protein Painting (CPP), a simple and fast method to infer structural information on protein conformation in cells with a quantitative protein footprinting technology. CPP monitors the conformational landscape of the 3D proteome in cells with high sensitivity and throughput. A key advantage of CPP is its' ability to quantitatively compare the 3D proteomes between different experimental conditions and to discover significant changes in the protein conformations. We detail how to perform a successful CPP experiment, the factors to consider before performing the experiment, and how to interpret the results.

Automated proteomic sample preparation: The key component for high throughput and quantitative mass spectrometry analysis.

Sample preparation for mass spectrometry-based proteomics has many tedious and time-consuming steps that can introduce analytical errors. In particular, the steps around the proteolytic digestion of protein samples are prone to inconsistency. One route for reliable sample processing is the development and optimization of a workflow utilizing an automated liquid handling workstation. Diligent assessment of the sample type, protocol design, reagents, and incubation conditions can significantly improve the speed and consistency of preparation. When combining robust liquid chromatography-mass spectrometry with either discovery or targeted methods, automated sample preparation facilitates increased throughput and reproducible quantitation of biomarker candidates. These improvements in analysis are also essential to process the large patient cohorts necessary to validate a candidate biomarker for potential clinical use. This article reviews the steps in the workflow, optimization strategies, and known applications in clinical, pharmaceutical, and research fields that demonstrate the broad utility for improved automation of sample preparation in the proteomic field.

iTRAQ-based proteomic analysis of Deinococcus radiodurans in response to 12C6+ heavy ion irradiation.

BACKGROUND: Deinococcus radiodurans (D. radiodurans) is best known for its extreme resistance to diverse environmental stress factors, including ionizing radiation (IR), ultraviolet (UV) irradiation, oxidative stress, and high temperatures. Robust DNA repair system and antioxidant system have been demonstrated to contribute to extreme resistance in D. radiodurans. However, practically all studies on the mechanism underlying D. radiodurans's extraordinary resistance relied on the treated strain during the post-treatment recovery lag phase to identify the key elements involved. The direct gene or protein changes of D. radiodurans after stress have not yet been characterized. RESULTS: In this study, we performed a proteomics profiling on D. radiodurans right after the heavy ion irradiation treatment, to discover the altered proteins that were quickly responsive to IR in D. radiodurans. Our study found that D. radiodurans shown exceptional resistance to 12C6+ heavy ion irradiation, in contrast to Escherichia coli (E.coli) strains. By using iTRAQ (Isobaric Tags for Relative and Absolute Quantitation)-based quantitative mass spectrometry analysis, the kinetics of proteome changes induced by various dosages of 12C6+ heavy ion irradiation were mapped. The results revealed that 452 proteins were differentially expressed under heavy ion irradiation, with the majority of proteins being upregulated, indicating the upregulation of functional categories of translation, TCA cycle (Tricarboxylic Acid cycle), and antioxidation regulation under heavy ion irradiation. CONCLUSIONS: This study shows how D. radiodurans reacts to exposure to 12C6+ heavy ion irradiation in terms of its overall protein expression profile. Most importantly, comparing the proteome profiling of D. radiodurans directly after heavy ion irradiation with research on the post-irradiation recovery phase would potentially provide a better understanding of mechanisms underlying the extreme radioresistance in D. radiodurans.