scholarly journals The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics

2019 ◽  
Vol 2 (2) ◽  
pp. 46 ◽  
Author(s):  
Suguru Saito ◽  
Yoshitoshi Hirao ◽  
Ali F. Quadery ◽  
Bo Xu ◽  
Amr Elguoshy ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Biomarker Discovery ◽  
Trypsin Digestion ◽  
Mass Spectrometry Analysis ◽  
Protein Preparation ◽  
Spectrometry Analysis ◽  
Urine Proteomics ◽  
Stability And Accuracy ◽  
Urine Proteins ◽  
Digestion Step

The sample condition is an important factor in urine proteomics with stability and accuracy. However, a general protocol of urine protein preparation in mass spectrometry analysis has not yet been established. Here, we proposed a workflow for optimized sample preparation based on methanol/chloroform (M/C) precipitation and in-solution trypsin digestion in LC-MS/MS-based urine proteomics. The urine proteins prepared by M/C precipitation showed around 80% of the protein recovery rate. The samples showed the largest number of identified proteins, which were over 1000 on average compared with other precipitation methods in LC-MS/MS-based urine proteomics. For further improvement of the workflow, the essences were arranged in protein dissolving and trypsin digestion step for the extraction of urine proteins. Addition of Ethylene diamine tetraacetic acid (EDTA) dramatically enhanced the dissolution of protein and promoted the trypsin activity in the digestion step because the treatment increased the number of identified proteins with less missed cleavage sites. Eventually, an optimized workflow was established by a well-organized strategy for daily use in the LC-MS/MS-based urine proteomics. The workflow will be of great help for several aims based on urine proteomics approaches, such as diagnosis and biomarker discovery.

scholarly journals Mass spectrometry analysis of the diversity of Aβ peptides: difficulties and future perspectives for AD biomarker discovery

2018 ◽  
Vol 15 (10) ◽  
pp. 773-775 ◽  
Author(s):  
Natalia V. Zakharova ◽  
Anna E. Bugrova ◽  
Alexey S. Kononikhin ◽  
Maria I. Indeykina ◽  
Igor A. Popov ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Biomarker Discovery ◽  
Mass Spectrometry Analysis ◽  
Future Perspectives ◽  
Aβ Peptides ◽  
Spectrometry Analysis

Sensitivity of mass spectrometry analysis depends on the shape of the filtration unit used for filter aided sample preparation (FASP)

PROTEOMICS ◽  
2016 ◽  
Vol 16 (13) ◽  
pp. 1852-1857 ◽  
Author(s):  
Joanna Lipecka ◽  
Cerina Chhuon ◽  
Matthieu Bourderioux ◽  
Marie-Andrée Bessard ◽  
Peter van Endert ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Filtration Unit

scholarly journals Gel Electrophoresis/Electroelution Sorting Fractionator combined with Filter Aided Sample preparation (FASP) for deep proteomic analysis

2021 ◽  
Author(s):  
Yassel Ramos ◽  
Alexis Almeida ◽  
Jenis Carpio ◽  
Arielis Rodríguez-Ulloa ◽  
Yasser Perera ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Identification ◽  
Protein Extraction ◽  
Complex Mixture ◽  
Fold Increase ◽  
Mass Spectrometry Analysis ◽  
Protein Fractionation ◽  
Spectrometry Analysis ◽  
Sds Page

AbstractSample preparation and protein fractionation are important issues in proteomic studies in spite of the technological achievements on protein mass spectrometry. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is SDS-PAGE-based protein fractionation due to its extraordinary resolution and the effectiveness of SDS as a solubilizing agent. Its main limitation lies in the poor recovery of the gel-trapped proteins, where protein electro-elution is the most successful approach to overcome this drawback. We created a device to separate complex mixture of proteins and peptides (named “GEES fractionator”) that is based on the continuous Gel Electrophoresis/Electro-elution Sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electro-eluted to the solution containing wells. The performance of the device was studied for SDS-PAGE-based protein fractionation in terms of reproducibility, protein recovery and loading capacity. In the SDS-free PAGE setup, complex peptide mixtures can also be fractionated. More than 11 700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the GEES fractionator combined with the Filter Aided Sample Preparation (FASP) method and mass spectrometry analysis. GEES-based proteome characterization shows a 1.7 fold increase in the number of identified proteins compared to the unfractionated sample analysis. Proteins involved in the co-regulated transcription activity, as well as cancer related pathways such as apoptosis signaling, P53 and RAS pathways are more represented in the protein identification output of GEES-based fractionation approaches.

scholarly journals Urine Proteomics Differentiate Primary Thrombotic Antiphospholipid Syndrome From Obstetric Antiphospholipid Syndrome

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhuochao Zhou ◽  
Yijun You ◽  
Fan Wang ◽  
Yue Sun ◽  
Jialin Teng ◽  
...  
Keyword(s):  
Antiphospholipid Syndrome ◽  
Fetal Loss ◽  
Absolute Quantification ◽  
Mass Spectrometry Analysis ◽  
Response To Treatment ◽  
Enzyme Linked Immunosorbent Assay ◽  
Multisystem Disorder ◽  
Spectrometry Analysis ◽  
Urine Proteomics ◽  
Non Invasive

Antiphospholipid syndrome (APS) is a multisystem disorder characterized by thrombosis and/or recurrent fetal loss. This clinical phenotype heterogeneity may result in differences in response to treatment and prognosis. In this study, we aimed to identify primary thrombotic APS (TAPS) from primary obstetric APS (OAPS) using urine proteomics as a non-invasive method. Only patients with primary APS were enrolled in this study from 2016 to 2018 at a single clinical center in Shanghai. Urine samples from 15 patients with TAPS, 9 patients with OAPS, and 15 healthy controls (HCs) were collected and analyzed using isobaric tags for relative and absolute quantification (iTRAQ) labeling combined with liquid chromatography-tandem mass spectrometry analysis to identify differentially expressed proteins. Cluster analysis of urine proteomics identified differentiated proteins among the TAPS, OAPS, and HC groups. Urinary proteins were enriched in cytokine and cytokine receptor pathways. Representative secreted cytokines screened out (fold change >1.20, or <0.83, p<0.05) in these differentiated proteins were measured by enzyme-linked immunosorbent assay in a validation cohort. The results showed that the levels of C-X-C motif chemokine ligand 12 (CXCL12) were higher in the urine of patients with TAPS than in those with OAPS (p=0.035), while the levels of platelet-derived growth factor subunit B (PDGFB) were lower in patients with TAPS than in those with OAPS (p=0.041). In addition, correlation analysis showed that CXCL12 levels were positively correlated with immunoglobulin G anti-β2-glycoprotein I antibody (r=0.617, p=0.016). Our results demonstrated that urinary CXCL12 and PDGFB might serve as potential non-invasive markers to differentiate primary TAPS from primary OAPS.

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