In-solution Protein Digestion Research Articles

Evaluation of Serum Proteome Sample Preparation Methods to Support Clinical Proteomics Applications.

Serum contains several proteins that are associated with disease-related processes. Mass spectrometry (MS)-based proteomics approaches greatly facilitate serum protein biomarker development. However, the serum proteome complexity presents a technical challenge for the accurate, sensitive, and reproducible quantification of proteins by MS. Thus, efficient sample preparation methods are of critical importance for serum proteome analyses. In this study, we evaluated the technical performance of two serum proteome sample preparation methods using sera from patients with high-grade serous ovarian cancer and patients with benign nongynecological conditions with a goal of providing insight into their compatibility with clinical proteomics workflows. One method entailed the use of immobilized trypsin (SMART Digest Trypsin) with RapiGest SF, an acid-labile surfactant designed to enhance the in-solution enzymatic digestion of proteins. The other method incorporated a commercially available sample preparation kit, iST-BCT, which contains standardized reagents. Significantly higher protein sequence coverage, albeit with lower digestion efficiency, was obtained with the immobilized trypsin + RapiGest SF workflow, whereas the iST-BCT workflow was quicker and had marginally better reproducibility. Protein relative abundance analysis revealed that the serum proteomes clustered primarily by the sample processing workflow and secondarily by disease state. We conducted a time course study to determine whether differences in the relative abundance of diagnostic high-grade serous ovarian cancer serum protein biomarker candidates were biased according to the duration of enzymatic digestion. Our results highlight the importance of optimizing enzymatic digestion kinetics according to the peptide targets of interest while considering the sensitivity of the downstream analytical method utilized in clinical proteomics workflows designed to measure biomarkers.

Salivary protein homology between humans and dogs: Mass spectrometry-based proteomics analysis

ObjectiveThis benchmark study aimed to investigate sex-related differences based on the identification and characterization of the salivary proteome of healthy male and female dogs using mass spectrometry (MS) technique and a homology-driven approach to analyze salivary proteins in both human and dog species utilizing protein sequence alignment technique. MethodsUnstimulated whole saliva was collected from 10 healthy Beagles. After processing the samples and determining the total protein content, in-solution protein digestion was performed involving denaturation, reduction of disulfide bonds, alkylation, and removal of interfering compounds. Samples were analyzed using LC-ESI-MS/MS. ResultsLC-ESI-MS/MS analysis identified 327 and 341 unique proteins in male and female dog saliva, respectively, of which 318 (97.25 %) in male dogs and 326 (95.60 %) in female dogs were characterized. Abundant shared proteins included albumin, BPI fold-containing family A member 2, and VWFD domain-containing protein. A notable uncharacterized protein, VWFD domain-containing protein, was among the most abundant in both sexes. Comparative analysis of 69 abundant shared proteins indicated an upregulation of CES5A, EFHD, GC, IGHM, LOC100653049, KRT10, LCP1, PGD, TPI1 in male dogs, while LOC100855593 was upregulated in female dogs. In total, 84 % (n = 229/274) and 86 % (n = 235/275) salivary proteins identified in male and female dogs, respectively, were homologous to human proteins, with an overall homology of 86 % (n = 364/423), including 15 with 100 % homology. ConclusionThe study revealed clear differences in the salivary proteomics profile of healthy male and female dogs. However, most of the salivary proteins in both male and female dogs showed homology with human salivary proteins. Clinical relevanceThe identification of unique salivary proteome profiles in male and female dogs, coupled with substantial homology to human proteins, provides promising biomarkers for health assessment, highlighting its clinical significance for diagnostics and therapeutic exploration not only in veterinary and human dentistry, but across mammalian species.

Preparation of Hard Tissues Like Bone or Cartilage for Shotgun Mass Spectrometry Analysis of the Proteome.

Proteomic analyses of intervertebral discs (IVDs) reveal information for understanding the fundamentals of biological processes and pathogenesis but also provide insights for novel pharmaceutical development. Sensitive mass spectrometry techniques and bioinformatics have advanced the detection and identification of proteins from any sample. Due to the challenges of catastrophic sample-loss artifacts during hard-tissue extraction, however, many researchers have omitted the cartilage endplates of IVDs for protein extraction, analyzing only the cellular components of the annulus fibrosus and/or nucleus pulposus. The full proteomic picture of IVDs is compromised without extracting proteins from intact IVDs. Here, we describe a novel preparation method using snap-freeze grinding, which allows for mechanical disruption and customized chemical lysis of hard tissues such as bone or cartilage. This method replaces the time-consuming and insufficient conventional tissue homogenization methods. Sample loss and contamination could be minimized during proteolysis by using an in-solution protein digestion and desalting procedure. We demonstrate excellent proteome coverage with intact mouse IVDs by analyzing samples in a hybrid quadrupole time-of-flight tandem mass spectrometer. © 2021 Wiley Periodicals LLC.

Phosphoproteome Analysis in Immune Cell Signaling.

Immune cell signaling is largely regulated by protein phosphorylation. Stimulation of toll-like receptors (TLRs) by pathogen-associated ligands drives the cascade of immune response, which can be influenced by differences in phosphoprotein abundance. Therefore, the analysis of phosphorylation signatures at a global level is central to understanding the complex and integrated signaling in macrophages upon pathogen attack. Here, we describe a mass spectrometry-based approach to identify and quantify phosphoproteome changes in response to the stimulation of TLR2, TLR4, and TLR7 with immune-response inducing ligands in cultured immune cells. This review will focus on the TLR stimulation of mouse macrophages as an example; however, the technique is applicable to any immortalized immune cell and any soluble stimuli. The methodology includes protocols for metabolic labeling of immune cells (stable isotope labeling of amino acids in cell culture, i.e., SILAC); ligand-initiated stimulation of immune receptors followed by cell lysis; in-solution trypsin digestion of proteins and enrichment of the resulting peptide mix for collecting phosphopeptides, which are then analyzed by high-resolution LC-MS/MS (liquid-chromatography tandem mass spectrometry). © 2020 Wiley Periodicals LLC. Basic Protocol 1: SILAC labeling of mouse macrophages Basic Protocol 2: Stimulation, cell lysis and Western Blotting Basic Protocol 3: Trypsin digestion, fractionation and phosphopeptide enrichment Basic Protocol 4: Quantitative mass spectrometry Alternate Protocol: Culturing SILAC-labeled cells from frozen mouse macrophages cells.

Discrimination of meat from fur-producing and food-providing animals using mass spectrometry-based proteomics

Non-edible meat from fur-producing animals entering into meat consumption chain could pose a serious threat to public health. For the purpose of risk prevention and control of meat safety, in this study, marker peptides for discriminating non-edible meat of fur-producing animals (including fox, silver fox, blue fox, raccoon dog, ussuri raccoon dog, mink and American mink) from meat of food-providing animals (including pig, cattle, sheep and donkey) were explored by shot-gun proteomics and verified by target approach. Two mass spectrometry platforms combined with bioinformatic and chemometric tools were integratedly emloyed for method development. Meat samples were first subjected to in-solution protein digestion and the subsequently tryptic peptides were profiled and quantitated by ultra-high pressure liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF MS) with sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) mode. Candidate marker peptides screened by chemometric tools were further filtered for their biological specificity and detectability through bioinformatics analysis as well as multiple reaction monitoring (MRM) verification with UHPLC-triple quadrupole mass spectrometry (UHPLC-QQQ MS). As a result, 9 peptides, out of 104 candidates, were selected as markers for discriminating analysis, of which DQTLQEELAR was validated as primary indicator of non-edible meat from the concerned fur-producing animals. An MRM method based on the developed marker peptides for routine use was finally proposed for risk alarming of non-edible meat from fur-producing animals in food safety control.

Fabrication of immobilized enzyme reactors with pillar arrays into polydimethylsiloxane microchip

This paper demonstrates the design, efficiency and applicability of a simple and inexpensive microfluidic immobilized enzymatic reactor (IMER) for rapid protein digestion. The high surface-to-volume ratio (S/V) of the reactor was achieved by forming pillars in the channel. It was found that pillar arrays including dimensions of 40 μm × 40 μm as pillar diameter and interpillar distance can provide both relatively high S/V and flow rate in the PDMS chip, the fabrication of which was performed by means of soft lithography using average research laboratory infrastructure. CZE peptide maps of IMER-based digestions were compared to peptide maps obtained from standard in-solution digestion of proteins. The peak patterns of the electropherograms and the identified proteins were similar, however, digestion with the IMER requires less than 10 min, while in-solution digestion takes 16 h.

A Protein Decomplexation Strategy in Snake Venom Proteomics.

Snake venoms are complex mixtures of proteins and peptides that play vital roles in the survival of venomous snakes. As with their diverse pharmacological activities, snake venoms can be highly variable, hence the importance of understanding the compositional details of different snake venoms. However, profiling venom protein mixtures is challenging, in particular when dealing with the diversity of protein subtypes and their abundances. Here we described an optimized strategy combining a protein decomplexation method with in-solution trypsin digestion and mass spectrometry of snake venom proteins. The approach involves the integrated use of C18 reverse-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and nano-electrospray ionization tandem mass spectrometry (nano-ESI-LC-MS/MS).

Accurate quantification of β-hexosaminidase released from laboratory of allergic diseases 2 cells via liquid chromatography tandem mass spectrometry method

β-Hexosaminidase is one of the enzymes that is secreted from mast cells via antigen-induced degranulation and has frequently been used as an indicator of anaphylactic reactions. The main method for determining β-hexosaminidase is indirect, “substrate-based” and shows limitations. Hence, development of an accurate detecting method is particularly important and urgently needed. In this study we developed a liquid chromatography tandem mass spectrometry (LC–MS/MS) method for measuring β-hexosaminidase. Laboratory of allergic diseases 2 (LAD2) cells were stimulated with compound 48/80 (C48/80), the supernatant was collected and subjected to in-solution protein digestion; the obtained peptides were desalted, concentrated, separated and analyzed with an LC-tandem MS instrument. A peptide with the sequence “LAPGTIVEVWK” was selected for quantitative analysis, and four other peptides for qualitative research. The time-effect relationship curve was studied, and the results of the LC–MS/MS method were found to be almost consistent with those obtained via the conventional method. The method was then employed to measure β-hexosaminidase released from LAD2 cells stimulated with potential allergens, and the results showed that it can be applied to determine the potential allergenicity of drugs. This new method showed good specificity, high sensitivity and a wide application range. It could be used to evaluate allergic reactions, providing a guide for medication safety during clinical testing.

Temporal Analyses of Barley Malting Stages Using Shotgun Proteomics

Malt derived from barley malting is an essential raw material for beer brewing. In this study, we performed the first dynamic proteome survey during barley malting using a gel-free proteomics approach. This entailed in-solution tryptic digestion of precipitated proteins and analysis of peptides by nanoliquid chromatography coupled with tandem mass spectrometry. A total of 1418 proteins were identified from the five malting stages: Steep, 1, 3, 5 days after germination, and end of Kiln. About 900 proteins identified in this analysis were uncharacterized or predicted proteins. Integrating information from Uniprot90, Uniprot50, Pfam, Interpro databases and gene ontologies from EnsemblPlants, 796 of the predicted and uncharacterized proteins were provided functional annotations. Nearly 63% of the identified proteins were present during all the five time points suggesting a coordinated activation of major metabolic pathways during malting. GO enrichment analysis showed over-representation of proteins associated with translation, carbohydrate metabolism, and stress response. Analysis of variance of the spectral counts of proteins present in all the five malting stages identified 205 proteins with significant differences in their abundance. Proteins associated with carbohydrate metabolism especially enzyme activity regulation provide novel targets for malting barley breeding and for predicting malting quality.

Highly Efficient Exosome Isolation and Protein Analysis by an Integrated Nanomaterial-Based Platform.

Exosomes play important roles in mediating intercellular communication and regulating a variety of biological processes, but clear understanding of their functions and biogenesis has not been achieved, due to the high technical difficulties involved in analysis of small vesicular structures that contain a high proportion of membrane structures. Herein, we designed a novel approach to integrate two nanomaterials carrying varied surface properties, the hydrophilic, macroporous graphene foam (GF) and the amphiphilic periodic mesoporous organosilica (PMO), for efficient exosome isolation from human serum and effective protein profiling. The high specific surface area of GF, after modification with the antibody against the exosomal protein marker, CD63, allowed highly specific isolation of exosomes from complex biological samples with high recovery. Since the organic solvent, methanol, turned out to be the most effective lysis solution for releasing the exosomal proteins, the amphiphilic PMO was employed to rapidly recover the exosomal proteins, including the highly hydrophobic membrane proteins. The fine pores of PMO also acted as the nanoreactors to accelerate protein digestion that produced peptides subject to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A total of 334 proteins with 111 membrane proteins [31% of these contained >2 transmembrane domains (TMD)] were identified using the integrated GF/PMO platform. In contrast, with the commercial exosome isolation kit and the in-solution protein digestion method, only 151 proteins were found, with 28 being membrane proteins (only one contained three TMDs). Our results support that the integrated GF/PMO platform is of great value to facilitate the comprehensive characterization of exosomal proteins for better understanding of their functions and for identification of more exosome-based disease markers.

Serum proteomic pattern in female stress urinary incontinence.

The pathophysiology of Stress Urinary Incontinence (SUI) is poorly understood. The aim of this study was to identify the serum proteomic profile in patients with SUI and to replicate findings from a preceding study in which a significant difference in the urinary proteome was identified. Serum samples were collected from 38patients (19 SUI; 19 matched, continent controls). Sample preparation included serum albumin depletion, in-solution enzymatic digestion of proteins applying a combination of Gluc-C and trypsin and peptide separation using nano High Performance Liquid Chromatography. Label-free quantitation of peptides and proteins was performed after triplicate measurements using quadrupole time-of-flight mass spectrometry. Peptide identification was achieved by searching the Human SwissProt Database using Mascot and X!Tandem. Main outcome measure was the relative abundance of each detected protein in serum. Of 7012 identified proteins, 33 proteins were induced (detected in SUI, not in controls) and five proteins were depleted (detected in controls, not in SUI). All depleted proteins play a role in immune/DNA damage response. Induced proteins are involved in inflammatory response, response to cellular stress, coagulation and cytoskeleton stability/ motility. Plasma serine protease inhibitor (SERPINA5) was found induced and previously also showed a higher abundance in urine samples of SUI patients. Data are available via ProteomeXchange with identifier PXD008553.

Sample Clean-up Strategies for ESI Mass Spectrometry Applications in Bottom-up Proteomics: Trends from 2012 to 2016.

Bottom-up proteomics is a mass spectrometric (MS)-based approach for the characterization of peptides obtained from in-solution protein digestion. MS is favored over other methods for peptide and protein analysis because of its better sensitivity and high throughput. Inorganic ions and surfactants present in the sample or produced during tryptic digestion are detrimental in MS analysis and affect the proteome data, thus sample preparation for removal of these unwanted components has become essential. Here, we review 48 research papers on strategies for removal of salts and surfactants (in particular, SDS) prior to ESI-MS analysis in bottom-up proteomics from 2012 to 2016. The strategies were mostly based on SPE and membrane-based filter-aided sample preparation for salt and SDS removal, respectively. Some known limitations of SPE and filter-aided sample preparation procedures are that they can be time consuming, laborious, and require the use of organic solvents before a concentrated extract suitable for analysis is obtained. The development of faster analytical methods by reducing the sample preparation time and thereby, increasing sample throughput, and in a solvent-less and membrane-less operation, is a significant contribution to proteome research.

Proteomic Analysis of 3T3‐L1 Adipocytes Treated with Methylglyoxal; The Potential Impact of Dietary Sugar on Adipocyte Metabolism

Rising levels of obesity in the United States (US) and in nations adopting a Western diet are of concern due to the associated risk of chronic diseases. As an endocrine organ, adipose tissue regulates whole body immunity and energy homeostasis, but during obesity, disruptions in the normal metabolism of adipocytes, such as increased oxidative stress and reduced mitochondrial function, may contribute to disease development. Diets high in added sugar, particularly sucrose and high fructose corn syrup, may induce metabolic abnormalities in adipocytes by increasing the rate of glycolysis and the accumulation of glycolytic metabolites. Methylglyoxal (MG), a highly reactive metabolite of glycolysis, damages proteins and other cellular components. MG is found at higher concentrations in the serum and tissues of individuals with diabetes and is directly involved in the pathophysiology of diabetic complications, however, MG may have negative effects on cell metabolism before the onset of disease. Recent studies using animal models suggest MG accumulates in adipose tissue during high fructose feeding and impairs insulin signaling pathways. However, the specific effects of MG in adipocytes require further investigation. The objective of this study was to characterize changes in the proteome of mature adipocytes exposed to non‐toxic levels of MG. We treated fully differentiated 3T3‐L1 adipocytes with either MG or PBS as a control for 48 hours. Cells were collected, delipidated, and proteins precipitated. After denaturation, in‐solution protein digestion was performed with trypsin and the resulting peptide fragments were separated by liquid chromatography and scanned by mass spectrometry (MS), followed by additional MS/MS scanning. MS/MS data was analyzed with MaxQuant software and proteins identified by comparison with the Uniprot mouse database. The proteome of MG‐treated adipocytes was characterized by reduced expression of both mitochondria‐related proteins and proteins involved in endogenous antioxidant pathways. In addition, MG treatment altered the expression of proteins involved in several key metabolic pathways, including oxidative phosphorylation, the citric acid cycle, and pyruvate metabolism. We conclude that MG induces changes in adipocytes which may not only contribute to oxidative stress, but which may disrupt normal metabolic pathways. Though this may represent a temporary adaptation to MG exposure, over time, repeated such exposures may make adipocytes more vulnerable to the serious metabolic abnormalities known to be associated with obesity‐related diseases.Support or Funding InformationUSDA NIFA grant #2011‐38420‐20038, Core Pilot grant Indiana Clinical and Translational Sciences Institute #UL1TR001108

Large-Scale Filter-Aided Sample Preparation Method for the Analysis of the Ubiquitinome.

Protein ubiquitination regulates key cellular functions, including protein homeostasis and signal transduction. The digestion of ubiquitinated proteins with trypsin yields a glycine-glycine remnant bound to the modified lysine residue (K-ε-GG) that can be recognized by specific antibodies for immunoaffinity purification (IAP) and subsequent identification of ubiquitination sites by mass spectrometry. Previous ubiquitinome studies based on this strategy have consistently digested milligram amounts of protein as starting material using in-solution digestion protocols prior to K-ε-GG enrichment. Filter-aided sample preparation (FASP) surpasses in-solution protein digestion in cleavage efficiency, but its performance has thus far been shown for digestion of sample amounts on the order of micrograms. Because cleavage efficiency is pivotal in the generation of the K-ε-GG epitope recognized during IAP, here we developed a large-scale FASP method (LFASP) for digestion of milligram amounts of protein and evaluated its applicability to the study of the ubiquitinome. Our results demonstrate that LFASP-based tryptic digestion is efficient, robust, reproducible, and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and show a ∼3-fold reduction in the proportion of miscleaved peptides with the method presented here. Beyond ubiquitinome analysis, LFASP overcomes the general limitation in sample capacity of standard FASP-based protocols and can therefore be used for a variety of applications that demand a large(r) amount of starting material.

Targeted Proteomics for Multiplexed Verification of Markers of Colorectal Tumorigenesis

Targeted proteomic methods can accelerate the verification of multiple tumor marker candidates in large series of patient samples. We utilized the targeted approach known as selected/multiple reaction monitoring (S/MRM) to verify potential protein markers of colorectal adenoma identified by our group in previous transcriptomic and quantitative shotgun proteomic studies of a large cohort of precancerous colorectal lesions. We developed SRM assays to reproducibly detect and quantify 25 (62.5%) of the 40 selected proteins in an independent series of precancerous and cancerous tissue samples (19 adenoma/normal mucosa pairs; 17 adenocarcinoma/normal mucosa pairs). Twenty-three proteins were significantly up-regulated (n = 17) or downregulated (n = 6) in adenomas and/or adenocarcinomas, as compared with normal mucosa (linear fold changes ≥ ±1.3, adjusted p value <0.05). Most changes were observed in both tumor types (up-regulation of ANP32A, ANXA3, SORD, LDHA, LCN2, NCL, S100A11, SERPINB5, CDV3, OLFM4, and REG4; downregulation of ARF6 and PGM5), and a five-protein biomarker signature distinguished neoplastic tissue from normal mucosa with a maximum area under the receiver operating curve greater than 0.83. Other changes were specific for adenomas (PPA1 and PPA2 up-regulation; KCTD12 downregulation) or adenocarcinoma (ANP32B, G6PD, RCN1, and SET up-regulation; downregulated AKR1B1, APEX1, and PPA1). Some changes significantly correlated with a few patient- or tumor-related phenotypes. Twenty-two (96%) of the 23 proteins have a potential to be released from the tumors into the bloodstream, and their detectability in plasma has been previously reported. The proteins identified in this study expand the pool of biomarker candidates that can be used to develop a standardized precolonoscopy blood test for the early detection of colorectal tumors.

A Methodology for Comprehensive Analysis of Toll-Like Receptor Signaling in Macrophages.

A combination of high-throughput, multiplexed, quantitative methods with computational modeling and statistical approaches is required to obtain system-level understanding of biological function. Mass spectrometry (MS)-based proteomics has emerged as a preferred tool for the analysis of changes in protein abundance and their post-translational modification (PTM) levels at a global scale, comparable with genomic experiments and generating data suitable for use in mathematical modeling of signaling pathways. Here we describe a set of parallel bottom-up proteomic approaches to detect and quantify the global protein changes in total intracellular proteins, their phosphorylation, and the proteins released by active and passive secretion or shedding mechanisms (referred to as the secretome as reviewed in Makridakis and Vlahou, J Proteome 73:2291-2305, 2010) in response to the stimulation of Toll-like receptors (TLRs) with specific ligands in cultured macrophages. The method includes protocols for metabolic labeling of cells (SILAC: stable isotope labeling by amino acids in cell culture; Ong et al., Mol Cell Proteomics 1:376-386, 2002), ligand stimulation, cell lysis and media collection, in-gel and in-solution modification and digestion of proteins, phosphopeptide enrichment for phosphoproteomics, and LC-MS/MS analysis. With these methods, we can not only reliably quantify the relative changes in the TLR signaling components (Sjoelund et al., J Proteome Res 13:5185-5197, 2014) but also use the data as constraints for mathematical modeling.

Characterization of an immobilized enzyme reactor for on-line protein digestion

Despite the developments for faster liquid chromatographic and mass spectral detection techniques, the standard in-solution protein digestion for proteomic analyses has remained relatively unchanged. The typical in-solution trypsin protein digestion is usually the slowest part of the workflow, albeit one of the most important. The development of a highly efficient immobilized enzyme reactor (IMER) with rapid performance for on-line protein digestion would greatly decrease the analysis time involved in a proteomic workflow. Presented here is the development of a silica based IMER for on-line protein digestion, which produced rapid digestions in the presence of organic mobile phase for both model proteins and a complex sample consisting of the insoluble portion of a yeast cell lysate. Protein sequence coverage and identifications evaluated between the IMER and in-solution digestions were comparable. Overall, for a yeast cell lysate with only a 10s volumetric residence time on-column, the IMER identified 507 proteins while the in-solution digestion identified 490. There were no significant differences observed based on identified protein’s molecular weight or isoelectric point between the two digestion methods. Implementation of the IMER into the proteomic workflow provided similar protein identification results, automation for sample analysis, and reduced the analysis time by 15h.

Changes in the Proteome of Xylem Sap in Brassica oleracea in Response to Fusarium oxysporum Stress

Fusarium oxysporum f.sp. conlutinans (Foc) is a serious root-invading and xylem-colonizing fungus that causes yellowing in Brassica oleracea. To comprehensively understand the interaction between F. oxysporum and B. oleracea, composition of the xylem sap proteome of the non-infected and Foc-infected plants was investigated in both resistant and susceptible cultivars using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-solution digestion of xylem sap proteins. Whole genome sequencing of Foc was carried out and generated a predicted Foc protein database. The predicted Foc protein database was then combined with the public B. oleracea and B. rapa protein databases downloaded from Uniprot and used for protein identification. About 200 plant proteins were identified in the xylem sap of susceptible and resistant plants. Comparison between the non-infected and Foc-infected samples revealed that Foc infection causes changes to the protein composition in B. oleracea xylem sap where repressed proteins accounted for a greater proportion than those of induced in both the susceptible and resistant reactions. The analysis on the proteins with concentration change > = 2-fold indicated a large portion of up- and down-regulated proteins were those acting on carbohydrates. Proteins with leucine-rich repeats and legume lectin domains were mainly induced in both resistant and susceptible system, so was the case of thaumatins. Twenty-five Foc proteins were identified in the infected xylem sap and 10 of them were cysteine-containing secreted small proteins that are good candidates for virulence and/or avirulence effectors. The findings of differential response of protein contents in the xylem sap between the non-infected and Foc-infected samples as well as the Foc candidate effectors secreted in xylem provide valuable insights into B. oleracea-Foc interactions.

Profiling of urinary proteins in Karan Fries cows reveals more than 1550 proteins

Urine is a non-invasive source of biological fluid, which reflects the physiological status of the mammals. We have profiled the cow urinary proteome and analyzed its functional significance. The urine collected from three healthy cows was concentrated by diafiltration (DF) followed by protein extraction using three methods, namely methanol, acetone, and ammonium sulphate (AS) precipitation and Proteo Spin urine concentration kit (PS). The quality of the protein was assessed by two-dimensional gel electrophoresis (2DE). In-gel digestion method revealed more proteins (1191) in comparison to in-solution digestion method (541). Collectively, 938, 606 and 444 proteins were identified in LC-MS/MS after in-gel and in-solution tryptic digestion of proteins prepared by AS, PS and DF methods, respectively resulting in identification of a total of 1564 proteins. Gene ontology (GO) using Panther7.0 grouped the majority of the proteins into cytoplasmic (location), catalytic activity (function), and metabolism (biological processes), while Cytoscape grouped proteins into complement and coagulation cascades; protease inhibitor activity and wound healing. Functional significance of few selected proteins seems to play important role in their physiology. Comparative analysis with human urine revealed 315 overlapping proteins. This study reports for the first time evidence of more than 1550 proteins in urine of healthy cow donors.This article is part of a Special Issue entitled: Proteomics in India.

Quantitative proteomic analysis of serum proteins from oral cancer patients: comparison of two analytical methods.

Serum proteomic analysis can be a valuable approach for the discovery of protein biomarkers for early detection or monitoring of a disease. In this study, two analytical methods were compared for quantification of serum proteins in patients with oral cancer. In the first approach, we quantified serum proteins between oral squamous cell carcinoma (OSCC) and healthy control subjects by performing in-solution digestion of serum proteins, isobaric tags for relative and absolute quantification (iTRAQ) labeling of the resulting peptides, strong cation exchange (SCX) fractionation of labeled peptides and finally capillary liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis of the peptides. In the second approach, we first separated serum proteins with SDS-PAGE. The gel-separated proteins were then digested with trypsin and the resulting peptides were labeled with iTRAQ and analyzed with LC-MS/MS for protein quantification. A total of 319 serum proteins were quantified with the first proteomic approach whereas a total of 281 proteins were quantified by the second proteomic approach. Most of the proteins were identified and quantified by both approaches, suggesting that these methods are similarly effective for serum proteome analysis. This study provides compelling evidence that quantitative serum proteomic analysis of OSCC is a valuable approach for identifying differentially expressed proteins in cancer patients’ circulation systems that may be used as potential biomarkers for disease detection. Further validation in large oral cancer patient populations may lead to a simple and low invasive clinical tool for OSCC diagnosis or monitoring.