Tandem Mass Tag Labeling Research Articles

Celastrol exerts a neuroprotective effect by directly binding to HMGB1 protein in cerebral ischemia\u2013reperfusion

BackgroundCelastrol (cel) was one of the earliest isolated and identified chemical constituents of Tripterygium wilfordii Hook. f. Based on a cel probe (cel-p) that maintained the bioactivity of the parent compound, the targets of cel in cerebral ischemia–reperfusion (I/R) injury were comprehensively analyzed by a quantitative chemical proteomics method.MethodsWe constructed an oxygen–glucose deprivation (OGD) model in primary rat cortical neurons and a middle cerebral artery occlusion (MCAO) model in adult rats to detect the direct binding targets of cel in cerebral I/R. By combining various experimental methods, including tandem mass tag (TMT) labeling, mass spectrometry, and cellular thermal shift assay (CETSA), we revealed the targets to which cel directly bound to exert neuroprotective effects.ResultsWe found that cel inhibited the proinflammatory activity of high mobility group protein 1 (HMGB1) by directly binding to it and then blocking the binding of HMGB1 to its inflammatory receptors in the microenvironment of ischemia and hypoxia. In addition, cel rescued neurons from OGD injury in vitro and decreased cerebral infarction in vivo by targeting HSP70 and NF-κB p65.ConclusionCel exhibited neuroprotective and anti-inflammatory effects by targeting HSP70 and NF-κB p65 and directly binding to HMGB1 in cerebral I/R injury.

Decellularized tendon matrix membranes prevent post-surgical tendon adhesion and promote functional repair

Adhesion often occurs after tendon injury, and results in sliding disorder and movement limitation with no ideal solution for it in clinic. In this study, an anti-adhesion membrane, i.e., decellularized tendon matrix (DTM) for tendon is successfully prepared by an optimized tendon decellularization method from homologous extracellular matrix. Microsection technology has been used to optimize the method of decellularization in order to better preserve the bioactive components in tissues and reduce the chemical reagent residues on the premise of effective decellularization with relatively shorter time and less reagents for decellularization. The physic-chemical properties and biological functions of DTM are evaluated, and high-throughput and high-precision tandem mass tags (TMT) labeling proteomics technology is used to analyze protein components of DTM, which may provide the scientific support for application of the innovative product. In vitro biosafety tests show that DTM not only is non-toxic but also promote cell proliferation. Subcutaneous implantation test confirms that DTM is completely degraded after 12 weeks and there is no obvious inflammatory reaction. The results of Achilles tendon repair in rabbits show that DTM can not only prevent tendon adhesion but also improve the quality of tendon repair, which demonstrates its tremendous application potential. Statement of SignificanceThere is no ideal solution for adhesion after tendon injury. In this study, a dense tendon anti-adhesion membrane (DTM) was successfully prepared from homologous extracellular matrix (ECM). This DTM could effectively retain bioactive ingredients, and prevent adhesion as well as improve the quality of tendon repair in vivo. An optimized decellularization method was used which could effectively decellularize tendon in a short time, better preserve bioactive components, and reduce reagent residues. For the first time, high-throughput and high-precision tandem mass tags (TMT) labeling proteomics technology was used to qualitatively and quantitatively analyze the protein composition of fresh tendon, acellular tendon and DTM, which provided not only scientific support for the application of DTM, but also comprehensive and accurate data support for related research of bovine tendons and decellularization.

Nanoscale Solid-Phase Isobaric Labeling for Multiplexed Quantitative Phosphoproteomics.

We established a workflow for highly sensitive multiplexed quantitative phosphoproteomics using a nanoscale solid-phase tandem mass tag (TMT) labeling reactor. Phosphopeptides were first enriched by titanium oxide chromatography and then labeled with isobaric TMT reagents in a StageTip packed with hydrophobic polymer-based sorbents. We found that TMT-labeled singly phosphorylated peptides tend to flow through the titanium oxide column. Therefore, TMT labeling should be performed after the enrichment step from tryptic peptides, resulting in the need for microscale reactions with small amounts of phosphopeptides. Using an optimized protocol for tens to hundreds of nanograms of phosphopeptides, we obtained a nearly 10-fold increase in sensitivity compared to the conventional solution-based TMT protocol. We demonstrate that this nanoscale phosphoproteomics protocol works for 50 μg of HeLa proteins treated with selumetinib, and we successfully quantified the selumetinib-regulated phosphorylated sites on a proteome scale. The MS raw data files have been deposited with the ProteomeXchange Consortium via the jPOST partner repository (https://jpostdb.org) with the data set identifier PXD025536.

Comparative proteomic analysis of tomato (Solanum lycopersicum L.) shoots reveals crosstalk between strigolactone and auxin

As one of the main vegetable crops cultivated in the world, the tomato has advantages of high yield and economic benefits, and plays an important role in promoting farmers' income and social and economic growth. However, lateral branches during the growth process of tomato consume considerable nutrients and reduce the yield of tomato. Phytohormones such as strigolactone and auxin can inhibit the formation of lateral branches. However, the mechanism of their interaction is not particularly clear. To better understand the effects of exogenous strigolactone and auxin on tomato, proteome analyses of tomato shoots treated with exogenous GR24 and indole acetic acid were performed using an integrated approach involving tandem mass tag (TMT) labeling and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). We identified 6685 proteins, of which 5822 contained quantitative information. Many differentially expressed proteins (DEPs) were found in different comparisons, including 415, 148, and 130 DEPs in GR24 vs mock, IAA vs mock, and GR24 + IAA vs mock comparisons, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that ‘photosynthesis - antenna proteins’ were significantly enriched in three treatments. Our data can help reveal the interaction between strigolactone and auxin in tomato seedlings.

Abstract 1577: Caloric restriction mimetics as adjuvant to immune checkpoint inhibitors for treatment of melanoma

Abstract Immune checkpoint inhibitors (ICIs) have revolutionized melanoma therapy. Still, the 5-year survival for advanced melanoma is only 50%. This leads to two questions: what makes some tumors less responsive to ICIs, and what can be done to increase responsiveness? Prior research suggests that hypoxia in the tumor microenvironment (TME) decreases the efficacy of ICIs, and metformin, an antihyperglycemic and caloric restriction mimetic (CRM), is able to reverse this condition. We hypothesize that other CRMs will also increase responsiveness to ICIs through hypoxia, and potentially other mechanisms. We chose three CRMs to study alongside metformin: hydroxycitrate, resveratrol, and sulforaphane. To identify pathways and understand more about CRMs effect on tumor hypoxia, we performed a proteomics experiment on B16F10 murine melanoma cells treated with all four CRMs. Proteomics analysis was done with a tandem mass tag labeling. To determine effect of caloric restriction mimetics on immunotherapy efficacy, B16F10 cells will be injected subcutaneously in immunocompetent male and female C57BL/6J mice, separated into four groups: 1) no treatment control, 2) ICI therapy only, 3) CRM therapy only, 4) ICI + CRM combination. Our preliminary data shows that, like metformin, additional CRMs decrease oxidative phosphorylation in cancer cells. This supports the hypothesis that the other CRMs may function similarly to metformin in the TME. We started testing the effect of four CRMs on tumor growth and hypoxia in an animal model. Our ultimate goal is to improve responsiveness to immune checkpoint inhibitors in patients with metastatic melanoma. Citation Format: Lauren C. Morehead, Brian S. Koss, Katherine F. Wallis, Alan J. Tackett, Isabelle Racine Miousse. Caloric restriction mimetics as adjuvant to immune checkpoint inhibitors for treatment of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1577.

Characterizing the proteome of bullous pemphigoid blister fluid utilizing tandem mass tag labeling coupled with LC-MS/MS.

Bullous pemphigoid is an autoimmune blistering disease caused by autoantibodies against components of the cutaneous basement membrane zone. Autoantibodies lead to complement-dependent and -independent inflammation and blistering. Blister fluid is a valuable biologic resource, as it provides insight into both systemic and local microenvironment responses. Here, we utilized liquid chromatography with tandem mass spectrometry to characterize the bullous pemphigoid blister fluid proteome. We then depleted exosomes to better understand the exosomal versus non-exosomal proteome. We identified 339 proteins in the blister fluid of bullous pemphigoid patients. Gene ontology demonstrated enrichment of several key biologic processes including innate immune response, neutrophil degranulation, platelet degranulation, and complement activation. Exosome depletion resulted in a significant decrease in normalized reporter intensities of 192 proteins, consistent with our observation of a large number of exosomal proteins found in the blister fluid. We then compared the bullous pemphigoid blister fluid proteome to prior proteomic datasets in suction blister fluid, snake bites, and thermal burns, identifying 76 proteins unique to bullous pemphigoid. These include major basic protein, eosinophil peroxidase, galectin-10, and the immunoglobulin epsilon heavy constant region, consistent with tissue eosinophilia. We lastly validated several previously reported blister fluid exosomal components. Blister fluid in bullous pemphigoid contains a mixture of numerous biologic processes. While many of these processes are shared with blistering from alternative causes, we have identified several notable features unique to bullous pemphigoid.

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation.

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. The workflow is adaptable across many sample types, including cultured cells (e.g., mammalian, prokaryotic) and whole tissues (e.g., heart, lung, muscle), which are initially lysed/homogenized and with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol-affinity resin, which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, the labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves the understanding of the role of redox-dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

Evaluation and Optimization of High-Field Asymmetric Waveform Ion-Mobility Spectrometry for Multiplexed Quantitative Site-Specific N-Glycoproteomics.

The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has been shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified immunoglobulin M (IgM) protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size, and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 methods provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass spectrometry-based qualitative and quantitative N-glycoproteomics.

Proteomics Analysis of the Spinal Dorsal Horn in Diabetic Painful Neuropathy Rats With Electroacupuncture Treatment.

BackgroundClinical evidence demonstrates that electro-acupuncture (EA) of the Zu sanli (ST36) and Shen shu (BL23) acupoints is effective in relieving diabetic painful neuropathy (DPN); however, the underlying molecular mechanism requires further investigation, including the protein molecules associated with EA’s effects on DPN.MethodsSprague-Dawley adult male rats (n =36) were randomly assigned into control, DPN, and EA groups (n=12 each). After four weeks of EA treatment, response to mechanical pain and fasting blood glucose were analyzed. A tandem mass tag (TMT) labeling approach coupled with liquid chromatography with tandem mass spectrometry was used to identify potential biomarkers in the spinal dorsal horn. Further, proteomics analysis was used to quantify differentially expressed proteins (DEPs), and gene ontology, KEGG pathways, cluster, and string protein network interaction analyses conducted to explore the main protein targets of EA.ResultsCompared with the DPN model group, the mechanical pain threshold was significantly increased, while the fasting blood glucose levels were clearly decreased in EA group rats. Proteomics analysis was used to quantify 5393 proteins, and DEPs were chosen for further analyses, based on a threshold of 1.2-fold difference in expression level (P < 0.05) compared with control groups. Relative to the control group, 169 down-regulated and 474 up-regulated proteins were identified in the DPN group, while 107 and 328 proteins were up- and down-regulated in the EA treatment group compared with the DPN group. Bioinformatics analysis suggested that levels of proteins involved in oxidative stress injury regulation were dramatically altered during the EA effects on DPN.ConclusionsOur results provide the valuable protein biomarkers, which facilitates unique mechanistic insights into the DPN pathogenesis and EA analgesic, antioxidant stress and hypoglycemic effect.

TMT-based quantitative proteomics analysis reveals the key proteins related with the differentiation process of goat intramuscular adipocytes

BackgroundIntramuscular adipocytes differentiation is a complex process, which is regulated by various transcription factor, protein factor regulators and signal transduction pathways. However, the proteins and signal pathways that regulates goat intramuscular adipocytes differentiation remains unclear.ResultIn this study, based on nanoscale liquid chromatography mass spectrometry analysis (LC-MS/MS), the tandem mass tag (TMT) labeling analysis was used to investigate the differentially abundant proteins (DAPs) related with the differentiation process of goat intramuscular adipocytes. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment and protein-protein interaction network analyses were performed for the characterization of the identified DAPs. The candidate proteins were verified by parallel reaction monitoring analysis. As a result, a total of 123 proteins, 70 upregulation proteins and 53 downregulation proteins, were identified as DAPs which may be related with the differentiation process of goat intramuscular adipocytes. Furthermore, the cholesterol metabolism pathway, glucagon signaling pathway and glycolysis / gluconeogenesis pathway were noticed that may be the important signal pathways for goat Intramuscular adipocytes differentiation.ConclusionsBy proteomic comparison between goat intramuscular preadipocytes (P_IMA) and intramuscular adipocytes (IMA), we identified a series protein that might play important role in the goat intramuscular fat differentiation, such as SRSF10, CSRP3, APOH, PPP3R1, CRTC2, FOS, SERPINE1 and AIF1L, could serve as candidates for further elucidate the molecular mechanism of IMF differentiation in goats.

Probing activation‐driven changes in coagulation factor IX by mass spectrometry

BackgroundActivated factor IX (FIXa) is an inefficient enzyme that needs activated factor VIII (FVIII) for full activity. Recently, we identified a network of FVIII‐driven changes in FIXa employing hydrogen‐deuterium eXchange mass spectrometry (HDX‐MS). Some changes also occurred in active‐site inhibited FIXa, but others were not cofactor‐driven, in particular those within the 220‐loop (in chymotrypsin numbering). ObjectiveThe aim of this work is to better understand the zymogen‐to‐enzyme transition in FIX, with specific focus on substrate‐driven changes at the catalytic site. MethodsFootprinting mass spectrometry by HDX and Tandem‐Mass Tags (TMT) labelling were used to explore changes occurring upon the conversion from FIX into FIXa. Mutagenesis and kinetic studies served to assess the role of the 220‐loop. ResultsHDX‐MS displayed remarkably few differences between FIX and FIXa. In comparison with FIX, FIXa did exhibit decreased deuterium uptake at the N‐terminus region. This was more prominent when the FIXa active site was occupied by an irreversible inhibitor. TMT‐labelling showed that the N‐terminus is largely protected from labelling, and that inhibitor binding increases protection to a minor extent. Occupation of the active site also reduced deuterium uptake within the 220‐loop backbone. Mutagenesis within the 220‐loop revealed that a putative H‐bond network contributes to FIXa activity. TMT labeling of the N‐terminus suggested that these 220‐loop variants are more zymogen‐like than wild‐type FIXa. ConclusionIn the absence of cofactor and substrate, FIXa is predominantly zymogen‐like. Stabilization in its enzyme‐like form involves, apart from FVIII‐binding, also interplay between the 220‐loop, N‐terminus, and the substrate binding site.

Tandem Mass Tag-Based Quantitative Proteomic Analysis of Chicken Bursa of Fabricius Infected With Reticuloendotheliosis Virus.

Reticuloendotheliosis virus (REV) is a type C avian retrovirus that causes immunosuppression, dwarf syndrome, and lymphoma in infected hosts. In this study, we used tandem mass tag (TMT) labeling and liquid chromatography–tandem mass spectrometry (LC-MS/MS) to characterize protein alterations in chicken bursa of Fabricius, before and after REV infection at 7, 14, 21, and 28 days. Our data showed that 1,127, 999, 910, and 1,138 differentially expressed proteins were significantly altered at 7, 14, 21, and 28 days after REV infection, respectively. Morphological analysis showed that REV infection reduced in cortical lymphocytes, bursal follicle atrophy, and nuclear damage. Bioinformatics analysis indicated these proteins were mainly involved with immune responses, energy metabolism, cellular processes, biological regulation, metabolic processes, response to stimuli, and multicellular organismal process. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway cluster analysis showed that post-infection, proteins were enriched in the cell cycle, Wnt signaling, antigen processing and presentation, cytokine receptor interaction, adenosine 3′,5′-cyclic monophosphate signaling pathway, and NF-κB signaling. In addition, we observed that peroxiredoxin 4 (PRDX4), peroxiredoxin 6 (PRDX6), glutathione peroxidase 3 (GPX3), catalase (CAT), and peroxidasin (PXDN) were involved in oxidative stress. Some heat shock protein (HSP) family members such as HSPH1, DNAJA4, HSPA8, and HSPA4L also changed significantly after REV infection. These findings help clarify interactions between REV and the host and provides mechanistic insights on REV-induced host immunosuppression.

A Tandem Mass Tags (TMTs) labeling approach highlights differences between the shoot proteome of two Arabidopsis thaliana ecotypes, Col-0 and Ws.

Arabidopsis has become a powerful model to study morphogenesis, plant growth, development but also plant response to environmental conditions. Over 1000 Arabidopsis genomes are available and show natural genetic variations. Among them, the main reference accessions Wassilewskija (Ws) and Columbia (Col-0), originally growing at contrasted altitudes and temperatures, are widely studied, but data contributing to their molecular phenotyping are still scarce. A global quantitative proteomics approach using isobaric stable isotope labeling (Tandem Mass Tags, TMT) was performed on Ws and Col-0. Plants have been hydroponically grown at 16 h/8 h (light/dark cycle) at 23°C day/19°C night for three weeks. A TMT labeling of the proteins extracted from their shoots has been performed and showed a differential pattern of protein abundance between them. These results have allowed identifying several proteins families possibly involved in the differential responses observed for Ws and Col-0 during plant development and upon environmental changes. In particular, Ws and Col-0 mainly differ in photosynthesis, cell wall-related proteins, plant defense/stress, ROS scavenging enzymes/redox homeostasis and DNA/RNA binding/transcription/translation/protein folding.

TMT-based proteomic and bioinformatic analyses of human granulosa cells from obese and normal-weight female subjects

BackgroundIncreasing evidence supports a relationship between obesity and either infertility or subfertility in women. Most previous omics studies were focused on determining if the serum and follicular fluid expression profiles of subjects afflicted with both obesity-related infertility and polycystic ovary syndrome (PCOS) are different than those in normal healthy controls. As granulosa cells (GCs) are essential for oocyte development and fertility, we determined here if the protein expression profiles in the GCs from obese subjects are different than those in their normal-weight counterpart.MethodsGC samples were collected from obese female subjects (n = 14) and normal-weight female subjects (n = 12) who were infertile and underwent in vitro fertilization (IVF) treatment due to tubal pathology. A quantitative approach including tandem mass tag labeling and liquid chromatography tandem mass spectrometry (TMT) was employed to identify differentially expressed proteins. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were then conducted to interrogate the functions and pathways of identified proteins. Clinical, hormonal, and biochemical parameters were also analyzed in both groups.ResultsA total of 228 differentially expressed proteins were noted, including 138 that were upregulated whereas 90 others were downregulated. Significant pathways and GO terms associated with protein expression changes were also identified, especially within the mitochondrial electron transport chain. The levels of free fatty acids in both the serum and follicular fluid of obese subjects were significantly higher than those in matched normal-weight subjects.ConclusionsIn GCs obtained from obese subjects, their mitochondria were damaged and the endoplasmic reticulum stress response was accompanied by dysregulated hormonal synthesis whereas none of these changes occurred in normal-weight subjects. These alterations may be related to the high FFA and TG levels detected in human follicular fluid.

Common Reference-Based Tandem Mass Tag Multiplexing for the Relative Quantification of Peptides: Design and Application to Degradome Analysis of Diphtheria Toxoid.

Currently, animal tests are being used to confirm the potency and lack of toxicity of toxoid vaccines. In a consistency approach, animal tests could be replaced if production consistency (compared to known good products) can be proven in a panel of in vitro assays. By mimicking the in vivo antigen processing in a simplified in vitro approach, it may be possible to distinguish aberrant products from good products. To demonstrate this, heat-exposed diphtheria toxoid was subjected to partial digestion by cathepsin S (an endoprotease involved in antigen processing), and the peptide formation/degradation kinetics were mapped for various heated toxoids. To overcome the limitations associated with the very large number of samples, we used common reference-based tandem mass tag (TMT) labeling. Instead of using one label per condition with direct comparison between the set of labels, we compared multiple labeled samples to a common reference (a pooled sample containing an aliquot of each condition). In this method, the number of samples is not limited by the number of unique TMT labels. This TMT multiplexing strategy allows for a 15-fold reduction of analysis time while retaining the reliability advantage of TMT labeling over label-free quantification. The formation of the most important peptides could be followed over time and compared among several conditions. The changes in enzymatic degradation kinetics of diphtheria toxoid revealed several suitable candidate peptides for use in a quality control assay that can distinguish structurally aberrant diphtheria toxoid from compliant toxoids.

Proteomics Landscape of Host-Pathogen Interaction in Acinetobacter baumannii Infected Mouse Lung.

Acinetobacter baumannii is an important pathogen of nosocomial infection worldwide, which can primarily cause pneumonia, bloodstream infection, and urinary tract infection. The increasing drug resistance rate of A. baumannii and the slow development of new antibacterial drugs brought great challenges for clinical treatment. Host immunity is crucial to the defense of A. baumannii infection, and understanding the mechanisms of immune response can facilitate the development of new therapeutic strategies. To characterize the system-level changes of host proteome in immune response, we used tandem mass tag (TMT) labeling quantitative proteomics to compare the proteome changes of lungs from A. baumannii infected mice with control mice 6 h after infection. A total of 6,218 proteins were identified in which 6,172 could be quantified. With threshold p < 0.05 and relative expression fold change > 1.2 or < 0.83, we found 120 differentially expressed proteins. Bioinformatics analysis showed that differentially expressed proteins after infection were associated with receptor recognition, NADPH oxidase (NOX) activation and antimicrobial peptides. These differentially expressed proteins were involved in the pathways including leukocyte transendothelial migration, phagocyte, neutrophil degranulation, and antimicrobial peptides. In conclusion, our study showed proteome changes in mouse lung tissue due to A. baumannii infection and suggested the important roles of NOX, neutrophils, and antimicrobial peptides in host response. Our results provide a potential list of protein candidates for the further study of host-bacteria interaction in A. baumannii infection. Data are available via ProteomeXchange with identifier PXD020640.

Assessment of TMT Labeling Efficiency in Large-Scale Quantitative Proteomics: The Critical Effect of Sample pH.

Isobaric labeling via tandem mass tag (TMT) reagents enables sample multiplexing prior to LC–MS/MS, facilitating high-throughput large-scale quantitative proteomics. Consistent and efficient labeling reactions are essential to achieve robust quantification; therefore, embedded in our clinical proteomic protocol is a quality control (QC) sample that contains a small aliquot from each sample within a TMT set, referred to as “Mixing QC.” This Mixing QC enables the detection of TMT labeling issues by LC–MS/MS before combining the full samples to allow for salvaging of poor TMT labeling reactions. While TMT labeling is a valuable tool, factors leading to poor reactions are not fully studied. We observed that relabeling does not necessarily rescue TMT reactions and that peptide samples sometimes remained acidic after resuspending in 50 mM HEPES buffer (pH 8.5), which coincided with low labeling efficiency (LE) and relatively low median reporter ion intensities (MRIIs). To obtain a more resilient TMT labeling procedure, we investigated LE, reporter ion missingness, the ratio of mean TMT set MRII to individual channel MRII, and the distribution of log 2 reporter ion ratios of Mixing QC samples. We discovered that sample pH is a critical factor in LE, and increasing the buffer concentration in poorly labeled samples before relabeling resulted in the successful rescue of TMT labeling reactions. Moreover, resuspending peptides in 500 mM HEPES buffer for TMT labeling resulted in consistently higher LE and lower missing data. By better controlling the sample pH for labeling and implementing multiple methods for assessing labeling quality before combining samples, we demonstrate that robust TMT labeling for large-scale quantitative studies is achievable.

Proteomic profiling of ovine milk after grading up.

We have previously bred Chinese local dairy sheep through grading up with local Small-Tailed Han (STH) sheep as female parent and DairyMeade (DM) sheep as male parent. In this research communication we characterize the whey protein profile of STH sheep and their offspring (F1, F2) to reveal physiological differences and variation in milk traits. A total of 1032 whey proteins were identified through tandem mass tag labeling (TMT) proteome profiling. Three proteins were significantly differentially abundant between F1 and STH milk, six between F2 and STH milk and five between F1 and F2 milk. In terms of differential changes between generations, WASHC4 and CUTA of F1 and Ig-like domain-containing protein of F2 milk were dominant whey proteins. Overall, the results showed that the whey protein profiles of different generations varied little. The crossbreeds of STH and DM sheep would be suitable for the development of the Chinese local sheep milk industry, and the F2 may be a better population for sheep milk production.

Tianlongkechuanling Inhibits Pulmonary Fibrosis Through Down-Regulation of Arginase-Ornithine Pathway.

Background: Pulmonary Fibrosis (PF) is an interstitial lung disease characterized by excessive accumulation of extracellular matrix in the lungs, which disrupts the structure and gas exchange of the alveoli. There are only two approved therapies for PF, nintedanib (Nib) and pirfenidone. Therefore, the use of Chinese medicine for PF is attracting attention. Tianlongkechuanling (TL) is an effective Chinese formula that has been applied clinically to alleviate PF, which can enhance lung function and quality of life. Purpose: The potential effects and specific mechanisms of TL have not been fully explored, yet. In the present study, proteomics was performed to explore the therapeutic protein targets of TL on Bleomycin (BLM)-induced Pulmonary Fibrosis. Method: BLM-induced PF mice models were established. Hematoxylineosin staining and Masson staining were used to analyze histopathological changes and collagen deposition. To screen the differential proteins expression between the Control, BLM, BLM + TL and BLM + Nib (BLM + nintedanib) groups, quantitative proteomics was performed using tandem mass tag (TMT) labeling with nanoLC-MS/MS [nano liquid chromatographymass spectrometry]). Changes in the profiles of the expressed proteins were analyzed using the bioinformatics tools Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The protein–protein interactions (PPI) were established by STRING. Expressions of α-smooth muscle actin (α-SMA), Collagen I (Col1a1), Fibronectin (Fn1) and enzymes in arginase-ornithine pathway were detected by Western blot or RT-PCR. Result: TL treatments significantly ameliorated BLM-induced collagen deposition in lung tissues. Moreover, TL can inhibit the protein expressions of α-SMA and the mRNA expressions of Col1a1 and Fn1. Using TMT technology, we observed 253 differentially expressed proteins related to PPI networks and involved different KEGG pathways. Arginase-ornithine pathway is highly significant. The expression of arginase1 (Arg1), carbamoyltransferase (OTC), carbamoy-phosphate synthase (CPS1), argininosuccinate synthase (ASS1), ornithine aminotransferase (OAT) argininosuccinate lyase (ASL) and inducible nitric oxide synthase (iNOS) was significantly decreased after TL treatments. Conclusion: Administration of TL in BLM-induced mice resulted in decreasing pulmonary fibrosis. Our findings propose that the down regulation of arginase-ornithine pathway expression with the reduction of arginase biosynthesis is a central mechanism and potential treatment for pulmonary fibrosis with the prevention of TL.