scholarly journals Shotgun Proteomics and Biomarker Discovery

2002 ◽  
Vol 18 (2) ◽  
pp. 99-105 ◽  
Author(s):  
W. Hayes McDonald ◽  
John R. Yates
Keyword(s):  
Large Scale ◽  
Protein Identification ◽  
Biomarker Discovery ◽  
Dynamic Range ◽  
Shotgun Proteomics ◽  
Sequence Information ◽  
Protein Biomarkers ◽  
Post Translational Modifications ◽  
Multidimensional Protein Identification Technology ◽  
Shotgun Approach

Coupling large-scale sequencing projects with the amino acid sequence information that can be gleaned from tandem mass spectrometry (MS/MS) has made it much easier to analyze complex mixtures of proteins. The limits of this “shotgun” approach, in which the protein mixture is proteolytically digested before separation, can be further expanded by separating the resulting mixture of peptides prior to MS/MS analysis. Both single dimensional high pressure liquid chromatography (LC) and multidimensional LC (LC/LC) can be directly interfaced with the mass spectrometer to allow for automated collection of tremendous quantities of data. While there is no single technique that addresses all proteomic challenges, the shotgun approaches, especially LC/LC-MS/MS-based techniques such as MudPIT (multidimensional protein identification technology), show advantages over gel-based techniques in speed, sensitivity, scope of analysis, and dynamic range. Advances in the ability to quantitate differences between samples and to detect for an array of post-translational modifications allow for the discovery of classes of protein biomarkers that were previously unassailable.

scholarly journals Integrating Genetics and the Plasma Proteome to Predict the Risk of Type 2 Diabetes

2020 ◽  
Vol 20 (11) ◽  
Author(s):  
Julia Carrasco Zanini ◽  
Maik Pietzner ◽  
Claudia Langenberg
Keyword(s):  
Type 2 Diabetes ◽  
Drug Targets ◽  
Information Transfer ◽  
Large Scale ◽  
Biomarker Discovery ◽  
Plasma Proteome ◽  
Protein Biomarkers ◽  
Genetic Studies ◽  
Proteomic Data

Abstract Purpose of the Review Proteins are the central layer of information transfer from genome to phenome and represent the largest class of drug targets. We review recent advances in high-throughput technologies that provide comprehensive, scalable profiling of the plasma proteome with the potential to improve prediction and mechanistic understanding of type 2 diabetes (T2D). Recent Findings Technological and analytical advancements have enabled identification of novel protein biomarkers and signatures that help to address challenges of existing approaches to predict and screen for T2D. Genetic studies have so far revealed putative causal roles for only few of the proteins that have been linked to T2D, but ongoing large-scale genetic studies of the plasma proteome will help to address this and increase our understanding of aetiological pathways and mechanisms leading to diabetes. Summary Studies of the human plasma proteome have started to elucidate its potential for T2D prediction and biomarker discovery. Future studies integrating genomic and proteomic data will provide opportunities to prioritise drug targets and identify pathways linking genetic predisposition to T2D development.

scholarly journals Mapping the Melanoma Plasma Proteome (MPP) Using Single-Shot Proteomics Interfaced with the WiMT Database

Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6224
Author(s):  
Natália Almeida ◽  
Jimmy Rodriguez ◽  
Indira Pla Parada ◽  
Yasset Perez-Riverol ◽  
Nicole Woldmar ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Large Scale ◽  
Protein Identification ◽  
Dynamic Range ◽  
Protein Sequences ◽  
Single Shot ◽  
Protein Database ◽  
Plasma Analysis ◽  
Novel Proteins ◽  
Simple Sample Preparation

Plasma analysis by mass spectrometry-based proteomics remains a challenge due to its large dynamic range of 10 orders in magnitude. We created a methodology for protein identification known as Wise MS Transfer (WiMT). Melanoma plasma samples from biobank archives were directly analyzed using simple sample preparation. WiMT is based on MS1 features between several MS runs together with custom protein databases for ID generation. This entails a multi-level dynamic protein database with different immunodepletion strategies by applying single-shot proteomics. The highest number of melanoma plasma proteins from undepleted and unfractionated plasma was reported, mapping >1200 proteins from >10,000 protein sequences with confirmed significance scoring. Of these, more than 660 proteins were annotated by WiMT from the resulting ~5800 protein sequences. We could verify 4000 proteins by MS1t analysis from HeLA extracts. The WiMT platform provided an output in which 12 previously well-known candidate markers were identified. We also identified low-abundant proteins with functions related to (i) cell signaling, (ii) immune system regulators, and (iii) proteins regulating folding, sorting, and degradation, as well as (iv) vesicular transport proteins. WiMT holds the potential for use in large-scale screening studies with simple sample preparation, and can lead to the discovery of novel proteins with key melanoma disease functions.

scholarly journals Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids

2021 ◽  
Author(s):  
Angela Mc Ardle ◽  
Aleksandra Binek, ◽  
Annie Moradian ◽  
Blandine Chazarin Orgel ◽  
Alejandro Rivas ◽  
...  
Keyword(s):  
High Throughput ◽  
Whole Blood ◽  
Large Scale ◽  
Dynamic Range ◽  
Profile Analysis ◽  
Protein Biomarkers ◽  
Throughput Analysis ◽  
Broad Dynamic Range ◽  
Plasma Fractions ◽  
Deep Profile

Background: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that allows flexibility for high and mid–throughput analysis for three blood–based proteomes: naive plasma, plasma depleted of the 14 most abundant proteins, and dried blood. Methods: Optimal conditions for sample preparation and DIA–MS analysis were established in plasma then automated and adapted for depleted plasma and whole blood. The MS workflow was modified to facilitate sensitive high–throughput or deep profile analysis with mid–throughput analysis. Analytical performance was evaluated from 5 complete workflows repeated over 3 days as well as a linearity analysis of a 5—6–point dilution curve. Result: Using our high-throughput workflow, 74%, 93%, 87% of peptides displayed an inter-day CV<30% in plasma, depleted plasma and whole blood. While the mid-throughput workflow had 67%, 90%, 78% of peptides in plasma, depleted plasma and whole blood meeting the CV<30% standard. Lower limits of detection and quantitation were determined for proteins and peptides observed in each biofluid and workflow. Combining the analysis of both high–throughput plasma fractions exceeded the number of reliably identified proteins for individual biofluids in the mid–throughput workflows. Conclusion: The workflow established here allowed for reliable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow on a large scale will facilitate the translation of candidate markers into clinical use.

scholarly journals Does Filter Aided Sample Preparation (FASP) Provide Sufficient Method Linearity for Quantitative Plant Shotgun Proteomics?

2021 ◽  
Author(s):  
Tatiana Leonova ◽  
Christian Ihling ◽  
Mohamad Saoud ◽  
Robert Rennert ◽  
Ludger A. Wessjohann ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dynamic Range ◽  
Proteome Analysis ◽  
Shotgun Proteomics ◽  
Specific Protein ◽  
Protein Quantification ◽  
Protein Isolation ◽  
Isolation Technique ◽  
Bottle Neck ◽  
Shotgun Approach

Gel-free LC-based shotgun proteomics represents the current gold standard of proteome analysis due to its outstanding throughput, analytical resolution and reproducibility. Thereby, the efficiency of sample preparation, i.e., protein isolation, solubilization and proteolysis, directly affects the correctness and reliability of quantification, being therefore the bottle neck of shotgun proteomics. The desired performance of the sample preparation protocols can be achieved by application of detergents. However, these ultimately compromise reverse phase chromatographic separation and disrupt electrospray ionization. Filter aided sample preparation (FASP) represents an elegant approach to overcome these limitations. Although this method is comprehensively validated for cell proteomics, its applicability to plants and compatibility with plant-specific protein isolation protocols is still unknown, i.e., no data on linearity of underlying protein quantification methods for plant matrices is available. To fill this gap, we address here the potential of FASP in combination with two protein isolation protocols for quantitative analysis of pea (<i>Pisum sativum</i>) seed and <i>Arabidopsis thaliana</i> leaf proteomes by the shotgun approach. For this, in comprehensive spiking experiments with bovine serum albumin (BSA), we evaluated the linear dynamic range (LDR) of protein quantification in the presence of plant matrices. Further, we addressed the interference of two different plant matrices in quantitative experiments, accomplished with two alternative sample preparation workflows in comparison to conventional FASP-based digestion of cell lysates, considered here as a reference. Our results indicate very good applicability of FASP to quantitative plant proteomics with an only limited impact of the protein isolation technique on the methods overall performance.<br>

scholarly journals Bayesian Mass Spectra Peak Alignment from Mass Charge Ratios

2008 ◽  
Vol 6 ◽  
pp. 117693510800600
Author(s):  
Junfeng Liu ◽  
Weichuan Yu ◽  
Baolin Wu ◽  
Hongyu Zhao
Keyword(s):  
Large Scale ◽  
Protein Identification ◽  
Biomarker Discovery ◽  
Severe Disease ◽  
Data Preprocessing ◽  
Tuning Parameter ◽  
Data Set ◽  
Experimental Calibration ◽  
Peak Alignment ◽  
Cross Validation Error

Proteomics studies based on mass spectrometry (MS) are gaining popular applications in biomedical research for protein identification/quantification and biomarker discovery, especially for potential early diagnosis and prognosis of severe disease before the occurrence of symptoms. However, MS data collected using current technologies are very noisy and appropriate data preprocessing is critical for successful applications of MS-based approaches. Among various data preprocessing steps, peak alignment from multiple spectra based on detected peak sample locations presents special statistical challenges when effective experimental calibration is not feasible due to relatively large peak location variation. To avoid intensive tuning parameter optimization, we propose a simple novel Bayesian algorithm “random grafting-pruning Markov chain Monte Carlo (RGPMCMC)” that can be applied to global MS peak alignment and to follow certain modelbased sample classification criterion for using aligned peaks to classify spectrum samples. The usefulness of our approach is demonstrated through simulation study by making extensive comparison with other algorithms in the literature. Its application to an ovarian cancer MALDI-MS data set achieves a smaller 10-fold cross validation error rate than other current large scale methodologies.

scholarly journals Does Filter Aided Sample Preparation (FASP) Provide Sufficient Method Linearity for Quantitative Plant Shotgun Proteomics?

2021 ◽  
Author(s):  
Tatiana Leonova ◽  
Christian Ihling ◽  
Mohamad Saoud ◽  
Robert Rennert ◽  
Ludger A. Wessjohann ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dynamic Range ◽  
Proteome Analysis ◽  
Shotgun Proteomics ◽  
Specific Protein ◽  
Protein Quantification ◽  
Protein Isolation ◽  
Isolation Technique ◽  
Bottle Neck ◽  
Shotgun Approach

Gel-free LC-based shotgun proteomics represents the current gold standard of proteome analysis due to its outstanding throughput, analytical resolution and reproducibility. Thereby, the efficiency of sample preparation, i.e., protein isolation, solubilization and proteolysis, directly affects the correctness and reliability of quantification, being therefore the bottle neck of shotgun proteomics. The desired performance of the sample preparation protocols can be achieved by application of detergents. However, these ultimately compromise reverse phase chromatographic separation and disrupt electrospray ionization. Filter aided sample preparation (FASP) represents an elegant approach to overcome these limitations. Although this method is comprehensively validated for cell proteomics, its applicability to plants and compatibility with plant-specific protein isolation protocols is still unknown, i.e., no data on linearity of underlying protein quantification methods for plant matrices is available. To fill this gap, we address here the potential of FASP in combination with two protein isolation protocols for quantitative analysis of pea (<i>Pisum sativum</i>) seed and <i>Arabidopsis thaliana</i> leaf proteomes by the shotgun approach. For this, in comprehensive spiking experiments with bovine serum albumin (BSA), we evaluated the linear dynamic range (LDR) of protein quantification in the presence of plant matrices. Further, we addressed the interference of two different plant matrices in quantitative experiments, accomplished with two alternative sample preparation workflows in comparison to conventional FASP-based digestion of cell lysates, considered here as a reference. Our results indicate very good applicability of FASP to quantitative plant proteomics with an only limited impact of the protein isolation technique on the methods overall performance.<br>

scholarly journals A37 EXPLORING THE PROTEOMICS DIFFERENCES IN CROHN’S DISEASE PATIENTS

2021 ◽  
Vol 4 (Supplement_1) ◽  
pp. 150-151
Author(s):  
L Nogueira de Almeida ◽  
B Mainoli ◽  
A K Filyk ◽  
S A Hirota ◽  
C Lu ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Protein Identification ◽  
Biomarker Discovery ◽  
Biological Significance ◽  
Shotgun Proteomics ◽  
Pathway Enrichment Analysis ◽  
Label Free ◽  
Serum Samples ◽  
Anti Inflammatory

Abstract Background Canada has the highest prevalence rate of Crohn’s disease (CD) in North America. In Alberta, the yearly cost of anti-inflammatory drugs can be more than $25,000 per person; however, half of the patients do not respond to medication. CD is characterized by lesions in the small intestine due to inflammation, promoting diarrhea and abdominal pain. Prolonged chronic inflammation results in fibrotic strictures that are resistant to anti-inflammatory therapies and promote narrowing of the luminal space that ultimately require surgery. Currently, there is no biomarker to distinguish between the inflammatory or stricturing phenotype. Aims AIM 1: Profile serum samples from CD patients using a label-free shotgun-proteomics. AIM 2: Identify signatures and biomarkers that distinguish inflammatory and fibrotic strictures using a bioinformatics approach. Methods Serum samples from 15 CD patients with strictures and 15 CD patients without strictures (inflammatory phenotype), as diagnosed by ultrasound imaging, were analyzed by a standard shotgun-proteomics approach. Briefly, 200 µg of serum proteins were processed in a label-free protocol in combination with the filter-aided sample preparation (FASP) method. Liquid chromatography-tandem mass spectrometry was performed on an Orbitrap Fusion Lumos. Protein identification was accomplished by MaxQuant at a 1% false-discovery rate. Statistical significance was determined by the MSstats package, in the R software. To identify the biological significance of disturbed pathways, it was characterized by the protein-protein interactions and pathway enrichment analysis using String-DB and Metascape. Results It was identified a statistically significant protein panel between the two phenotypes. Proteins identified in the strictured group include JAK1 (Tyrosine-protein kinase), CD5 antigen-like protein (regulates inflammatory gene expression in Th17 cells), and neogenin (cell adhesion). Of the inflammatory patients, there was a significant elevation of PFK/FBPase 2 (synthesis and degradation of fructose 2,6-bisphosphate), vinculin (cell-matrix adhesion) and MMP-16/MT3-MMP (matrix metalloproteinase). Conclusions The identification of a distinct signature between both phenotypes provide important biological information about the disease progression and are a good sign that a biomarker discovery platform will be capable to differentiate between inflammatory and fibrostenotic strictures from serum samples of CD patients. Funding Agencies CAG, CIHRNSERC

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