Label-free Quantitative Method Research Articles

Identification and quantification of gelatin by a high-resolution mass spectrometry-based label-free method

High-performance liquid chromatography (HPLC) and linear-ion trap/Orbitrap high-resolution mass spectrometry with selected ion monitoring (SIM) mode were used to create a label-free quantitative method for bovine gelatin as a representative mammalian gelatin. Various indexes including linear correlation coefficient, specificity, and reproducibility were used to evaluate this quantitative method via pure bovine gelatin as well as prepared edible films. After accurate mass and tandem mass spectrometry, seventeen marker peptides were detected and identified in pure bovine gelatin. Of these, seven marker peptides were confirmed to quantify pure bovine gelatin with good linearity (R2 ≥ 0.99 for calibration equations), specificity, and reproducibility. Moreover, with excellent quantitative results, two peptides with m/z of 824.91772+ and 1076.01782+ were shown to be useful as characteristic peptides for quantifying bovine gelatin in the edible film systems constituted by mixed gelatins with various ratios and food additives. Due to the simple operation process, low experiment costs, and excellent measurement capability, this label-free method with high-resolution mass spectrometry could be a promising method to quantify gelatin in the food industry.

Early urine proteome changes in the Walker-256 tail-vein injection rat model

Detection of cancer at its early stage is important for treatment. Urine, which is not regulated by homeostatic mechanisms, reflects early systemic changes throughout the whole body and can be used for the early detection of cancer. In this study, the Walker-256 tail-vein injection rat model was established to find whether the urine proteome could reflect early changes if tumor grown in lung. Urine samples from the control group (n = 7) and Walker-256 tail-vein injection group (n = 7) on days 2, 4, 6 and 9 were analyzed by label-free proteomic quantitative methods. On day 2, when lung tumor nodules did not appear, 62 differential proteins were identified. They were associated with epithelial cell differentiation, regulation of immune system processes and the classical complement activation pathway. On day 4, when lung tumor nodules appeared, 72 differential proteins were identified. They were associated with the innate immune response and positive regulation of phagocytosis. On day 6, when body weight began to decrease, 117 differential proteins were identified. On day 9, the identified 125 differential proteins were associated with the B cell receptor signaling pathway and the positive regulation of B cell activation. Our results indicate that (1) the urine proteome changed even on the second day after tail-vein injection of Walker-256 cells and that (2) compared to previous studies, the urine proteomes were different when the same cancer cells were grown in different organs.

Urine Proteome Changes in a TNBS-Induced Colitis Rat Model.

Urine is an important resource for biomarker research. Urine proteins reflect not only renal diseases but also changes in other organs in the body. However, urine has rarely been used to reflect on inflammatory bowel disease. In the present study, a trinitrobenzene sulfonic acid (TNBS)-induced colitis rat model is used to mimic the human inflammatory bowel disease Crohn's disease (CD). Urine samples are analyzed by label-free and TMT-labeled proteomic quantitative methods. 77 urinary proteins are significantly changed in the colitis rats compared with that in the controls. These proteins are further validated by parallel reaction monitoring (PRM) targeted proteomic quantitative methods. Based on the human protein tissue atlas, carbonic anhydrase 1, ribonuclease pancreatic gamma type, and neutral and basic amino acid transport protein rBAT are highly enriched in the gastrointestinal tract. Among the nine PRM-validated proteins, carbonic anhydrase 1, neutrophil collagenase, and neutrophil gelatinase associated lipocalin were previously reported as IBD-associated proteins (all exhibiting consistent trends with the observation herein), whereas the others are newly discovered by this study. The results provide valuable clues for future study of urine biomarker of inflammatory bowel disease and Crohn's disease.

Quantitative Analysis of Ubiquitinated Proteins in Human Pituitary and Pituitary Adenoma Tissues.

Protein ubiquitination is an important post-translational modification that is associated with multiple diseases, including pituitary adenomas (PAs). Protein ubiquitination profiling in human pituitary and PAs remains unknown. Here, we performed the first ubiquitination analysis with an anti-ubiquitin antibody (specific to K-ε-GG)-based label-free quantitative proteomics method and bioinformatics to investigate protein ubiquitination profiling between PA and control tissues. A total of 158 ubiquitinated sites and 142 ubiquitinated peptides in 108 proteins were identified, and five ubiquitination motifs were found. KEGG pathway network analysis of 108 ubiquitinated proteins identified four statistically significant signaling pathways, including PI3K-AKT signaling pathway, hippo signaling pathway, ribosome, and nucleotide excision repair. R software Gene Ontology (GO) analysis of 108 ubiquitinated proteins revealed that protein ubiquitination was involved in multiple biological processes, cellular components, and molecule functions. The randomly selected ubiquitinated 14-3-3 zeta/delta protein was further analyzed with Western blot, and it was found that upregulated 14-3-3 zeta/delta protein in nonfunctional PAs might be derived from the significantly decreased level of its ubiquitination compared to control pituitaries, which indicated a contribution of 14-3-3 zeta/delta protein to pituitary tumorigenesis. These findings provided the first ubiquitinated proteomic profiling and ubiquitination-involved signaling pathway networks in human PAs. This study offers new scientific evidence and basic data to elucidate the biological functions of ubiquitination in PAs, insights into its novel molecular mechanisms of pituitary tumorigenesis, and discovery of novel biomarkers and therapeutic targets for effective treatment of PAs.

Proteomic Analysis of Horseweed (Conyza canadensis) Subjected to Caprylic Acid Stress.

Caprylic acid (CAP) is anticipated to be a potential biocontrol herbicide in the control of weeds, however the molecular mechanism of how CAP affects weeds is poorly understood. Here, the physiological and biochemical (protein-level) changes in horseweed (Conyza canadensis L.) are studied under CAP treatment, with infrared gas analyzer and label-free quantitative proteomics methods. In total, 112 differentially-accumulated proteins (DAPs) (>1.5 fold change, p < 0.05) are present between treated horseweed and control samples, with 46 up-regulated and 66 down-regulated proteins. These DAPs are involved in 28 biochemical pathways, including photosynthesis pathways. In particular, six photosynthesis proteins show significant abundance changes in the CAP-treated horseweed. The qRT-PCR results confirm three of the six genes involved in photosynthesis. Moreover, by measuring photosynthesis characteristics, CAP was shown to decrease photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and the transpiration rate of horseweed. These results suggest that photosystem I is one of the main biological processes involved in the response of horseweed to CAP.

MALDI-TOF Characterization of Protein Expression Mutation During Morphological Changes of Bacteria Under the Impact of Antibiotics.

Antimicrobial resistance (AMR) is one of the most serious problems affecting public health and safety. When treated by antibiotics, bacteria usually experience changes in morphology that can lead to the development of AMR. In this work, we propose a strategy to study mutation in protein expression during morphological changes of bacteria under the impact of antibiotics. The study is focused on small proteins that can be detected by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which is nowadays widely used in hospitals for bacterial identification. We used a gradient microfluidic chip to observe the morphological changes of various bacteria under the impact of antibiotics. Differential peaks related to the morphological changes were first figured out by MALDI-TOF MS, then identified by considering the molecular weight of both candidate proteins and their tryptic digested peptides, and further validated by a liquid chromatography tandem mass spectrometry (LC-MS/MS) based label-free quantitative proteomic method. Specifically, carbapenem-resistant Klebsiella pneumoniae (CR-KP), super extended spectrum β-lactamases Escherichia coli, Vibrio parahemolyticus from South America prawns, and carbapenem-resistant Pseudomonas aeruginosa were used as model samples to illustrate the strategy. Eight proteins closely correlated with its morphological change were identified for CR-KP. Among the eight proteins, three, i.e., fimbrial subunit type 3, penicillin-binding protein activator LpoB, and 30S ribosomal protein S14, were further verified at the transcriptome level.

Application of Extracellular Vesicles Proteomics to Cardiovascular Disease: Guidelines, Data Analysis, and Future Perspectives.

Extracellular vesicles (EVs) are a heterogeneous population of vesicles composed of a lipid bilayer that carry a large repertoire of molecules including proteins, lipids, and nucleic acids. In this review, some guidelines for plasma-derived EVs isolation, characterization, and proteomic analysis, and the application of the above to cardiovascular disease (CVD) studies are provided. For EVs analysis, blood samples should be collected using a 21-gauge needle, preferably in citrate tubes, and plasma stored for up to 1 year at -80°, using a single freeze-thaw cycle. For proteomic applications, differential centrifugation (including ultracentrifugation steps) is a good option for EVs isolation. EVs characterization is done by transmission electron microscopy, particle enumeration techniques (nanoparticle-tracking analysis, dynamic light scattering), and flow cytometry. Regarding the proteomics strategy, a label-free and gel-free quantitative method is a good choice due to its accuracy and because it minimizes the amount of sample required for clinical applications. Besides the above, main EVs proteomic findings in cardiovascular-related diseases are presented and analyzed in this review, paying especial attention to overlapping results between studies. The latter might offer new insights into the clinical relevance and potential of novel EVs biomarkers identified to date in the context of CVD.

A Proteomic Approach for Understanding the Mechanisms of Delayed Corneal Wound Healing in Diabetic Keratopathy Using Diabetic Model Rat.

Diabetes mellitus is a widespread metabolic disorder, and long-term hyperglycemia in diabetics leads to diabetic keratopathy. In the present study, we used a shotgun liquid chromatography/mass spectrometry-based global proteomic approach using the cornea of streptozotocin-induced diabetic (STZ) rats to examine the mechanisms of delayed corneal wound healing in diabetic keratopathy. Applying a label-free quantitation method based on spectral counting, we identified 188 proteins that showed expression changes of >2.0-fold in the cornea of STZ rats. In particular, the level of lumican expression in the cornea of STZ rats was higher than that of the normal rats. In the cornea of the normal rat, the expression level of lumican was elevated during the wound healing process, and it returned to the same expression level as before cornea injury after the wound was healed completely. On the other hand, a high expression level of lumican in the cornea of STZ rats was still maintained even after the wound was healed completely. In addition, adhesion deficiency in corneal basal cells and Bowman’s membrane was observed in the STZ rat. Thus, abnormally overexpressed lumican may lead to adhesion deficiency in the cornea of STZ rats.

Label-free quantitative proteomic analysis revealed a positive effect of ectopic over-expression of PeaT1 from Alternaria tenuissima on rice (Oryza sativa) response to drought.

The protein elicitor PeaT1 was found in Alternaria tenuissima and exerted broad spectrum resistance in wheat, cotton, and rice. Recently, we found that overexpressing PeaT1 rice (OE) could enhance plant drought tolerance. Elucidating some elevated drought stress-related proteins and associated mechanisms is inevitable for improving drought tolerance in rice. In this study, combining a label-free quantitative proteomic method, multiple proteins were differentially accumulated in OE plants. Among these, a total of 57 significant changed proteins (including 32 up-regulated and 25 down-regulated) were mainly involved in metabolic, cellular, biological progress, and stress response. Using the RT-qPCR assay, 18 proteins' relative abundance was detected mostly consistent with the proteins abundance in proteomic data. Specially, proteins involved in abiotic stress, such as OsSKIPa and OsPP2C, which were significantly induced in early after dehydration treatment in transgenic rice, and the other stress response genes (prohibitin protein, PsbP protein, msrB Protein) also changed in PeaT1 OE lines. Taken together, these results suggested that these differential proteins would be helpful for understanding the functional molecular mechanism of PeaT1 in rice.

Imsnc761 and DDX6 synergistically suppress cell proliferation and promote apoptosis via p53 in testicular embryonal carcinoma cells.

Intermediate-sized non-coding RNAs (imsncRNAs) have been shown to play important regulatory roles in the development of several eukaryotic organisms. In the present research, we selected imsncRNA 761 (imsnc761) as a research target. Expression analyses in a previous study showed that imsnc761 was down-regulated in maturation-arrested testis tissues as compared with the level in normal controls. In the present study, we found that imsnc761 could interact with DEAD-box helicase 6 (DDX6) to induce NTERA-2 (NT2 (testicular embryonal carcinoma cell)) cell apoptosis and proliferation inhibition via the p53 pathway. This interaction between imsnc761 and DDX6 also inhibited mitochondrial function and specific gene transcription and translation. To facilitate further research, we used label-free quantitation method to analyze the associated differences in Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways and biological processes. This confirmed the changes in several specific pathways, which matched our molecular experimental results.

Comparative proteomics of cancer stem cells in osteosarcoma using ultra-high-performance liquid chromatography and Orbitrap Fusion mass spectrometer

Osteosarcoma is the most common malignant tumour found in bones, and it has a poor prognosis. For improved therapy, it is significant to have a deep understanding of the proteomics changes in the cancer stem cells (CSCs) of osteosarcoma. Therefore, a comparative proteomics approach based on ultra-high-performance liquid chromatography coupled to an Orbitrap Fusion mass spectrometer (UHPLC-Orbitrap Fusion MS) was established to investigate the key molecular changes between CSCs and non-CSCs in human osteosarcoma HOS cells. A proteomic analysis was performed on these samples and a total of more than 6600 proteins were identified in each run. Moreover, most of the correlation coefficients between three biological repeats were more than 0.9 in each group. That finding demonstrated not only that the reproducibility of the test is good but also that the stability of this MS is excellent. A label-free quantitative method was applied to analyse differentially expressed proteins. Using the criteria of greater than 1.5-fold changes and a p value < 0.05, 124 proteins were identified as being significantly different between HOS-CSCs and non-CSCs. A pathway analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed the potential molecular regulatory networks that may regulate CSCs. Selected differential α-actinin 4 (ACTN4) proteins were validated by Western blot assay. These findings enhance our understanding of the molecular changes in CSCs and may provide additional improvements in therapy for treating osteosarcoma. Moreover, the UHPLC-Orbitrap Fusion MS-based proteomics method is helpful in cancer research.

Bacterial Survival under Extreme UV Radiation: A Comparative Proteomics Study of Rhodobacter sp., Isolated from High Altitude Wetlands in Chile.

Salar de Huasco, defined as a polyextreme environment, is a high altitude saline wetland in the Chilean Altiplano (3800 m.a.s.l.), permanently exposed to the highest solar radiation doses registered in the world. We present here the first comparative proteomics study of a photoheterotrophic bacterium, Rhodobacter sp., isolated from this remote and hostile habitat. We developed an innovative experimental approach using different sources of radiation (in situ sunlight and UVB lamps), cut-off filters (Mylar, Lee filters) and a high-throughput, label-free quantitative proteomics method to comprehensively analyze the effect of seven spectral bands on protein regulation. A hierarchical cluster analysis of 40 common proteins revealed that all conditions containing the most damaging UVB radiation induced similar pattern of protein regulation compared with UVA and visible light spectral bands. Moreover, it appeared that the cellular adaptation of Rhodobacter sp. to osmotic stress encountered in the hypersaline environment from which it was originally isolated, might further a higher resistance to damaging UV radiation. Indeed, proteins involved in the synthesis and transport of key osmoprotectants, such as glycine betaine and inositol, were found in very high abundance under UV radiation compared to the dark control, suggesting the function of osmolytes as efficient reactive oxygen scavengers. Our study also revealed a RecA-independent response and a tightly regulated network of protein quality control involving proteases and chaperones to selectively degrade misfolded and/or damaged proteins.

Tag-Count Analysis of Large-Scale Proteomic Data.

Label-free quantitative methods are advantageous in bottom-up (shotgun) proteomics because they are robust and can easily be applied to different workflows without additional cost. Both label-based and label-free approaches are routinely applied to discovery-based proteomics experiments and are widely accepted as semiquantitative. Label-free quantitation approaches are segregated into two distinct approaches: peak-abundance-based approaches and spectral counting (SpC). Peak abundance approaches like MaxLFQ, which is integrated into the MaxQuant environment, require precursor peak alignment that is computationally intensive and cannot be routinely applied to low-resolution data. Not limited by these constraints, SpC approaches simply use the number of peptide identifications corresponding to a given protein as a measurement of protein abundance. We show here that spectral counts from multidimensional proteomic data sets have a mean-dispersion relationship that can be modeled in edgeR. Furthermore, by simulating spectral counts, we show that this approach can routinely be applied to large-scale discovery proteomics data sets to determine differential protein expression.

A label-free differential proteomics analysis reveals the effect of melatonin on promoting fruit ripening and anthocyanin accumulation upon postharvest in tomato.

To better understand the function of melatonin in tomato fruit ripening and quality improvement, a label-free quantitation method was used to investigate the proteins that differ between the control (CK) and 50 μm melatonin treatment (M50) fruits. Proteomics data identified 241 proteins that were significantly influenced by melatonin. These proteins were involved in several ripening-related pathways, including cell wall metabolism, oxidative phosphorylation, carbohydrate, and fatty acid metabolism. Moreover, the application of exogenous melatonin increased eight proteins that are related to anthocyanin accumulation during fruit ripening. Additionally, the affected protein levels correlated with the corresponding gene transcript levels. Further, the total anthocyanin content from M50 increased by 52%, 48%, and 50% at 5, 8, and 13 DAT (day after melatonin treatment), respectively. The melatonin-mediated promotion of fruit ripening and quality might be due to the altered proteins involved in processes associated with ripening. In this work, we indicated that a senescence-related protein was downregulated in the M50 fruit, while a cell apoptosis inhibitor (API5) protein was upregulated. In addition, peroxidases (POD9, POD12, peroxidase p7-like) and catalase (CAT3) significantly increased in the M50 fruits. Based on the previous studies and our data, we inferred that melatonin might be positively related to fruit ripening but negatively related to fruit senescence. This research provides insights into the physiological and molecular mechanisms underlying melatonin-mediated fruit ripening as well as the anthocyanin formation process in tomato fruit at the protein concentration level, and we reveal possible candidates for regulation of anthocyanin formation during fruit ripening.

Degradation of Swainsonine by the NADP-Dependent Alcohol Dehydrogenase A1R6C3 in Arthrobacter sp. HW08

Swainsonine is an indolizidine alkaloid that has been found in locoweeds and some fungi. Our previous study demonstrated that Arthrobacter sp. HW08 or its crude enzyme extract could degrade swainsonie efficiently. However, the mechanism of swainsonine degradation in bacteria remains unclear. In this study, we used label-free quantitative proteomics method based on liquid chromatography-electrospray ionization-tandem mass spectrometry to dissect the mechanism of swainsonine biodegradation by Arthrobacter sp. HW08. The results showed that 129 differentially expressed proteins were relevant to swainsonine degradation. These differentially expressed proteins were mostly related to the biological process of metabolism and the molecular function of catalytic activity. Among the 129 differentially expressed proteins, putative sugar phosphate isomerase/epimerase A1R5X7, Acetyl-CoA acetyltransferase A0JZ95, and nicotinamide adenine dinucleotide phosphate (NADP)-dependent alcohol dehydrogenase A1R6C3 were found to contribute to the swainsonine degradation. Notably, NADP-dependent alcohol dehyrodgenase A1R6C3 appeared to play a major role in degrading swainsonine, but not as much as Arthrobacter sp. HW08 did. Collectively, our findings here provide insights to understand the mechanism of swainsonine degradation in bacteria.

Comparative shotgun proteomic analysis of wild and domesticated Opuntia spp. species shows a metabolic adaptation through domestication

The Opuntia genus is widely distributed in America, but the highest richness of wild species are found in Mexico, as well as the most domesticated Opuntia ficus-indica, which is the most domesticated species and an important crop in agricultural economies of arid and semiarid areas worldwide. During domestication process, the Opuntia morphological characteristics were favored, such as less and smaller spines in cladodes and less seeds in fruits, but changes at molecular level are almost unknown. To obtain more insights about the Opuntia molecular changes through domestication, a shotgun proteomic analysis and database-dependent searches by homology was carried out. >1000 protein species were identified and by using a label-free quantitation method, the Opuntia proteomes were compared in order to identify differentially accumulated proteins among wild and domesticated species. Most of the changes were observed in glucose, secondary, and 1C metabolism, which correlate with the observed protein, fiber and phenolic compounds accumulation in Opuntia cladodes. Regulatory proteins, ribosomal proteins, and proteins related with response to stress were also observed in differential accumulation. These results provide new valuable data that will help to the understanding of the molecular changes of Opuntia species through domestication. Biological significanceOpuntia species are well adapted to dry and warm conditions in arid and semiarid regions worldwide, and they are highly productive plants showing considerable promises as an alternative food source. However, there is a gap regarding Opuntia molecular mechanisms that enable them to grow in extreme environmental conditions and how the domestication processes has changed them. In the present study, a shotgun analysis was carried out to characterize the proteomes of five Opuntia species selected by its domestication degree. Our results will help to a better understanding of proteomic features underlying the selection and specialization under evolution and domestication of Opuntia and will provide a platform for basic biology research and gene discovery.

Using PSEA-Quant for Protein Set Enrichment Analysis of Quantitative Mass Spectrometry-Based Proteomics.

PSEA-Quant analyzes quantitative mass spectrometry-based proteomics datasets to identify enrichments of annotations contained in repositories such as the Gene Ontology and Molecular Signature databases. It allows users to identify the annotations that are significantly enriched for reproducibly quantified high abundance proteins. PSEA-Quant is available on the Web and as a command-line tool. It is compatible with all label-free and isotopic labeling-based quantitative proteomics methods. This protocol describes how to use PSEA-Quant and interpret its output. The importance of each parameter as well as troubleshooting approaches are also discussed. © 2016 by John Wiley & Sons, Inc.

Integrated GlycoProteome Analyzer (I-GPA) for Automated Identification and Quantitation of Site-Specific N-Glycosylation.

Human glycoproteins exhibit enormous heterogeneity at each N-glycosite, but few studies have attempted to globally characterize the site-specific structural features. We have developed Integrated GlycoProteome Analyzer (I-GPA) including mapping system for complex N-glycoproteomes, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. Using an N-glycopeptide database that we constructed, we created novel scoring algorithms with decoy glycopeptides, where 95 N-glycopeptides from standard α1-acid glycoprotein were identified with 0% false positives, giving the same results as manual validation. Additionally automated label-free quantitation method was first developed that utilizes the combined intensity of top three isotope peaks at three highest MS spectral points. The efficiency of I-GPA was demonstrated by automatically identifying 619 site-specific N-glycopeptides with FDR ≤ 1%, and simultaneously quantifying 598 N-glycopeptides, from human plasma samples that are known to contain highly glycosylated proteins. Thus, I-GPA platform could make a major breakthrough in high-throughput mapping of complex N-glycoproteomes, which can be applied to biomarker discovery and ongoing global human proteome project.

Protein Quantitation of the Developing Cochlea Using Mass Spectrometry.

Mass spectrometry-based proteomics allows for the measurement of hundreds to thousands of proteins in a biological system. Additionally, mass spectrometry can also be used to quantify proteins and peptides. However, observing quantitative differences between biological systems using mass spectrometry-based proteomics can be challenging because it is critical to have a method that is fast, reproducible, and accurate. Therefore, to study differential protein expression in biological samples labeling or label-free quantitative methods can be used. Labeling methods have been widely used in quantitative proteomics, however label-free methods have become equally as popular and more preferred because they produce faster, cleaner, and simpler results. Here, we describe the methods by which proteins are isolated and identified from cochlear sensory epithelia tissues at different ages and quantitatively differentiated using label-free mass spectrometry.

Comparison of reliabilities of mass spectrometry-based label-free quantitation methods for histone post-translational modification analysis

Comparison of reliabilities of mass spectrometry-based label-free quantitation methods for histone post-translational modification analysis