Improved proteome coverage by using iTRAQ labelling and peptide OFFGEL fractionation

Background  

The development of mass spectrometric techniques and fractionation methods now allows the investigation of very complex protein mixtures ranging from subcellular structures to tissues. Nevertheless, this work is particularly difficult due to the wide dynamic range of protein concentration in eukaryotic tissues. In this paper, we present a shotgun method whereby the peptides are fractionated using OFFGEL electrophoresis after iTRAQ labelling.     

Optimization of iTRAQ labelling coupled to OFFGEL fractionation as a proteomic workflow to the analysis of microsomal proteins of Medicago truncatula roots

Background

Shotgun proteomics represents an attractive technical framework for the study of membrane proteins that are generally difficult to resolve using two-dimensional gel electrophoresis. The use of iTRAQ, a set of amine-specific isobaric tags, is currently the labelling method of choice allowing multiplexing of up to eight samples and the relative quantification of multiple peptides for each protein. Recently the hyphenation of different separation techniques with mass spectrometry was used in the analysis of iTRAQ labelled samples. OFFGEL electrophoresis has proved its effectiveness in isoelectric point-based peptide and protein separation in solution. Here we describe the first application of iTRAQ-OFFGEL-LC-MS/MS on microsomal proteins from plant material. The investigation of the iTRAQ labelling effect on peptide electrofocusing in OFFGEL fractionator was carried out on Medicago truncatula membrane protein digests.

Results

In-filter protein digestion, with easy recovery of a peptide fraction compatible with iTRAQ labelling, was successfully used in this study. The focusing quality in OFFGEL electrophoresis was maintained for iTRAQ labelled peptides with a higher than expected number of identified peptides in basic OFFGEL-fractions. We furthermore observed, by comparing the isoelectric point (pI) fractionation of unlabelled versus labelled samples, a non-negligible pI shifts mainly to higher values.

Conclusions

The present work describes a feasible and novel protocol for in-solution protein digestion in which the filter unit permits protein retention and buffer removal. The data demonstrates an impact of iTRAQ labelling on peptide electrofocusing behaviour in OFFGEL fractionation compared to their native counterpart by the induction of a substantial, generally basic pI shift. Explanations for the occasionally observed acidic shifts are likewise presented.     

Comparison of iTRAQ and SWATH in a clinical study with multiple time points

Background

Advances in mass spectrometry have accelerated biomarker discovery in many areas of medicine. The purpose of this study was to compare two mass spectrometry (MS) methods, isobaric tags for relative and absolute quantitation (iTRAQ) and sequential window acquisition of all theoretical fragment ion spectra (SWATH), for analytical efficiency in biomarker discovery when there are multiple methodological constraints such as limited sample size and several time points for each patient to be analyzed.

Methods

A total of 140 tear samples were collected from 28 glaucoma patients at 5 time points in a glaucoma drug switch study. Samples were analyzed with iTRAQ and SWATH methods using NanoLC-MSTOF mass spectrometry.

Results

We discovered that even though iTRAQ is faster than SWATH with respect to analysis time per sample, it loses in sensitivity, reliability and robustness. While SWATH analysis yielded complete data of 456 proteins in all samples, with iTRAQ we were able to quantify 477 proteins in total but on average only 125 proteins were quantified in a sample. 283 proteins were common in the datasets produced by the two methods. Repeatability of the methods was assessed by calculating percent relative standard deviation (% RSD) between replicate MS analyses: SWATH was more repeatable (56% of proteins?<?20% RSD), compared to iTRAQ (43% of proteins?<?20% RSD). Despite the overall benefits of SWATH, both methods showed less than 1 log fold change difference in the expression of 74% common proteins. In addition, comparison to MS/MS peptide results using 8 isotopically labeled peptide standards, SWATH and iTRAQ showed similar results in terms of accuracy. Moreover, both methods detected similar trends in a longitudinal analysis of protein expression of two known tear biomarkers.

Conclusions

Overall, we conclude that SWATH should be preferred for biomarker discovery studies when analyzing limited volumes of clinical samples collected at multiple time points.

Trial Registeration

The study was approved by the Ethics Committee at Tampere University Hospital and was registered in EU clinical trials register (EudraCT Number: 2010-021039-14).

     

Plasma protein profiling of Mild Cognitive Impairment and Alzheimer’s disease using iTRAQ quantitative proteomics

Background

With the promise of disease modifying treatments, there is a need for more specific diagnosis and prognosis of Alzheimer’s disease (AD) and mild cognitive impairment (MCI). Plasma biomarkers are likely to be utilised to increase diagnostic accuracy and specificity of AD and cognitive decline.

Methods

Isobaric tags (iTRAQ) and proteomic methods were used to identify potential plasma biomarkers of MCI and AD. Relative protein expression level changes were quantified in plasma of 411 cognitively normal subjects, 19 AD patients and 261 MCI patients. Plasma was pooled into 4 groups including normal control, AD, amnestic single and multiple domain MCI (aMCI), and nonamnestic single and multiple domain MCI (nMCI). Western-blotting was used to validate iTRAQ data. Integrated function and protein interactions were explored using WEB based bioinformatics tools (DAVID v6.7 and STRING v9.0).

Results

In at least two iTRAQ replicate experiments, 30 proteins were significantly dysregulated in MCI and AD plasma, relative to controls. These proteins included ApoA1, ApoB100, complement C3, C4b-binding protein, afamin, vitamin D-binding protein precursor, isoform 1 of Gelsolin actin regulator, Ig mμ chain C region (IGHM), histidine-rich glycoprotein and fibrinogen β and γ chains. Western-blotting confirmed that afamin was decreased and IGHM was increased in MCI and AD groups. Bioinformatics results indicated that these dysregulated proteins represented a diversity of biological processes, including acute inflammatory response, cholesterol transport and blood coagulation.

Conclusion

These findings demonstrate that expression level changes in multiple proteins are observed in MCI and AD plasma. Some of these, such as afamin and IGHM, may be candidate biomarkers for AD and the predementia condition of MCI.     

Aberrant Mucin5B expression in lung adenocarcinomas detected by iTRAQ labeling quantitative proteomics and immunohistochemistry

Background

Lung cancer is the number one cause of cancer-related deaths in the United States and worldwide. The complex protein changes and/or signature of protein expression in lung cancer, particularly in non-small cell lung cancer (NSCLC) has not been well defined. Although several studies have investigated the protein profile in lung cancers, the knowledge is far from complete. Among early studies, mucin5B (MUC5B) has been suggested to play an important role in the tumor progression. MUC5B is the major gel-forming mucin in the airway. In this study, we investigated the overall protein profile and MUC5B expression in lung adenocarcinomas, the most common type of NSCLCs.

Methods

Lung adenocarcinoma tissue in formalin-fixed paraffin-embedded (FFPE) blocks was collected and microdissected. Peptides from 8 tumors and 8 tumor-matched normal lung tissue were extracted and labeled with 8-channel iTRAQ reagents. The labeled peptides were identified and quantified by LC-MS/MS using an LTQ Orbitrap Velos mass spectrometer. MUC5B expression identified by iTRAQ labeling was further validated using immunohistochemistry (IHC) on tumor tissue microarray (TMA).

Results

A total of 1288 peptides from 210 proteins were identified and quantified in tumor tissues. Twenty-two proteins showed a greater than 1.5-fold differences between tumor and tumor-matched normal lung tissues. Fifteen proteins, including MUC5B, showed significant changes in tumor tissues. The aberrant expression of MUC5B was further identified in 71.1% of lung adenocarcinomas in the TMA.

Discussions

A subset of tumor-associated proteins was differentially expressed in lung adenocarcinomas. The differential expression of MUC5B in lung adenocarcinomas suggests its role as a potential biomarker in the detection of adenocarcinomas.     

Periostin identified as a potential biomarker of prostate cancer by iTRAQ-proteomics analysis of prostate biopsy

Background  

Proteomics may help us better understand the changes of multiple proteins involved in oncogenesis and progression of prostate cancer(PCa) and identify more diagnostic and prognostic biomarkers. The aim of this study was to screen biomarkers of PCa by the proteomics analysis using isobaric tags for relative and absolute quantification(iTRAQ).     

Quantitative Proteomic Analysis of Serum from Pregnant Women Carrying a Fetus with Conotruncal Heart Defect Using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Labeling

Objective

To identify differentially expressed proteins from serum of pregnant women carrying a conotruncal heart defects (CTD) fetus, using proteomic analysis.

Methods

The study was conducted using a nested case-control design. The 5473 maternal serum samples were collected at 14–18 weeks of gestation. The serum from 9 pregnant women carrying a CTD fetus, 10 with another CHD (ACHD) fetus, and 11 with a normal fetus were selected from the above samples, and analyzed by using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional liquid chromatography-tandem mass spectrometry(2D LC-MS/MS). The differentially expressed proteins identified by iTRAQ were further validated with Western blot.

Results

A total of 105 unique proteins present in the three groups were identified, and relative expression data were obtained for 92 of them with high confidence by employing the iTRAQ-based experiments. The downregulation of gelsolin in maternal serum of fetus with CTD was further verified by Western blot.

Conclusions

The identification of differentially expressed protein gelsolin in the serum of the pregnant women carrying a CTD fetus by using proteomic technology may be able to serve as a foundation to further explore the biomarker for detection of CTD fetus from the maternal serum.     

Altered proteolytic events in experimental autoimmune encephalomyelitis discovered by iTRAQ shotgun proteomics analysis of spinal cord

Background  

Abnormal activation of protease activities during experimental autoimmune encephalomyelitis (EAE) in rats, a rodent model of multiple sclerosis, have been implicated in either the direct destruction of myelin components or the intracellular signal transduction pathways that lead to lymphocyte infiltration, oligodendrocyte destruction, neuronal dysfunctions and axonal degeneration. The identification of changes in regulated proteolytic events during EAE is crucial for uncovering activated proteases that may underline the pathological features such as inflammation and demyelination. We searched for either non-tryptic or semi-tryptic peptides from a previous shotgun proteomics study using isobaric tags for relative and absolute quantification (iTRAQ) to compare the proteomes of normal and EAE rat lumbar spinal cords.     

Bacterial artificial chromosomes improve recombinant protein production in mammalian cells

Background  

The development of appropriate expression vectors for large scale protein production constitutes a critical step in recombinant protein production. The use of conventional expression vectors to obtain cell lines is a cumbersome procedure. Often, stable cell lines produce low protein yields and production is not stable over the time. These problems are due to silencing of randomly integrated expression vectors by the surrounding chromatin. To overcome these chromatin effects, we have employed a Bacterial Artificial Chromosome (BAC) as expression vector to obtain stable cell lines suitable for protein production.     

PreDisorder: ab initio sequence-based prediction of protein disordered regions

Background  

Disordered regions are segments of the protein chain which do not adopt stable structures. Such segments are often of interest because they have a close relationship with protein expression and functionality. As such, protein disorder prediction is important for protein structure prediction, structure determination and function annotation.     

ITRAQ-based quantitative proteomic analysis of processed <Emphasis Type="Italic">Euphorbia lathyris</Emphasis> L. for reducing the intestinal toxicity

Background

Euphorbia lathyris L., a Traditional Chinese medicine (TCM), is commonly used for the treatment of hydropsy, ascites, constipation, amenorrhea, and scabies. Semen Euphorbiae Pulveratum, which is another type of Euphorbia lathyris that is commonly used in TCM practice and is obtained by removing the oil from the seed that is called paozhi, has been known to ease diarrhea. Whereas, the mechanisms of reducing intestinal toxicity have not been clearly investigated yet.

Methods

In this study, the isobaric tags for relative and absolute quantitation (iTRAQ) in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic method was applied to investigate the effects of Euphorbia lathyris L. on the protein expression involved in intestinal metabolism, in order to illustrate the potential attenuated mechanism of Euphorbia lathyris L. processing. Differentially expressed proteins (DEPs) in the intestine after treated with Semen Euphorbiae (SE), Semen Euphorbiae Pulveratum (SEP) and Euphorbiae Factor 1 (EFL₁) were identified. The bioinformatics analysis including GO analysis, pathway analysis, and network analysis were done to analyze the key metabolic pathways underlying the attenuation mechanism through protein network in diarrhea. Western blot were performed to validate selected protein and the related pathways.

Results

A number of differentially expressed proteins that may be associated with intestinal inflammation were identified. They mainly constituted by part of the cell. The expression sites of them located within cells and organelles. G protein and Eph/Ephrin signal pathway were controlled jointly by SEP and SE. After processing, the extraction of SEP were mainly reflected in the process of cytoskeleton, glycolysis and gluconeogenesis.

Conclusions

These findings suggest that SE induced an inflammatory response, and activated the Interleukin signaling pathway, such as the Ang/Tie 2 and JAK2/ STAT signaling pathways, which may eventually contribute to injury result from intestinal inflammatory, while SEP could alleviate this injury by down-regulating STAT1 and activating Ang-4 that might reduce the inflammatory response. Our results demonstrated the importance of Ang-4 and STAT1 expression, which are the target proteins in the attenuated of SE after processing based on proteomic investigation. Thus iTRAQ might be a novel candidate method to study scientific connotation of hypothesis that the attenuated of SE after processing expressed lower toxicity from cellular levels.

     

Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase

Background  

Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

Adenosine infusion increases plasma levels of VEGF in humans

Background  

Many in vitro studies have shown that adenosine (Ado) can induce vascular endothelial growth factor (VEGF) mRNA and protein expression and stimulate endothelial proliferation. In the present study, we seek to determine whether Ado can increase circulating levels of VEGF protein in the intact human.