scholarly journals Improving the Identification and Coverage of Plant Transmembrane Proteins in Medicago Using Bottom–Up Proteomics

2020 ◽  
Vol 11 ◽  
Author(s):  
Han Chung Lee ◽  
Adam Carroll ◽  
Ben Crossett ◽  
Angela Connolly ◽  
Amani Batarseh ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cyanogen Bromide ◽  
Transmembrane Proteins ◽  
Environmental Cues ◽  
Signaling Proteins ◽  
Protein Cleavage ◽  
Peptide Fragments ◽  
Bottom Up ◽  
Complete Identification ◽  
Cleavage Method

Plant transmembrane proteins (TMPs) are essential for normal cellular homeostasis, nutrient exchange, and responses to environmental cues. Commonly used bottom–up proteomic approaches fail to identify a broad coverage of peptide fragments derived from TMPs. Here, we used mass spectrometry (MS) to compare the effectiveness of two solubilization and protein cleavage methods to identify shoot-derived TMPs from the legume Medicago. We compared a urea solubilization, trypsin Lys-C (UR-TLC) cleavage method to a formic acid solubilization, cyanogen bromide and trypsin Lys-C (FA-CTLC) cleavage method. We assessed the effectiveness of these methods by (i) comparing total protein identifications, (ii) determining how many TMPs were identified, and (iii) defining how many peptides incorporate all, or part, of transmembrane domains (TMD) sequences. The results show that the FA-CTLC method identified nine-fold more TMDs, and enriched more hydrophobic TMPs than the UR-TLC method. FA-CTLC identified more TMPs, particularly transporters, whereas UR-TLC preferentially identified TMPs with one TMD, particularly signaling proteins. The results suggest that combining plant membrane purification techniques with both the FA-CTLC and UR-TLC methods will achieve a more complete identification and coverage of TMPs.

scholarly journals Mass spectrometry‐based top‐down and bottom‐up approaches for proteomic analysis of the Moroccan Buthus occitanus scorpion venom

FEBS Open Bio ◽  
2021 ◽  
Author(s):  
Khadija Daoudi ◽  
Christian Malosse ◽  
Ayoub Lafnoune ◽  
Bouchra Darkaoui ◽  
Salma Chakir ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Proteomic Analysis ◽  
Scorpion Venom ◽  
Top Down ◽  
Bottom Up

scholarly journals Vicilin and legumin storage proteins are abundant in water and alkali soluble protein fractions of glandless cottonseed

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zhongqi He ◽  
Christopher P. Mattison ◽  
Dunhua Zhang ◽  
Casey C. Grimm
Keyword(s):  
Mass Spectrometry ◽  
Soluble Protein ◽  
Extraction Efficiency ◽  
Storage Proteins ◽  
Protein Composition ◽  
Protein Fractions ◽  
Peptide Fragments ◽  
2S Albumin ◽  
Cottonseed Protein ◽  
Glandless Cottonseed

AbstractIn this work, we sequentially extracted water (CSPw)- and alkali (CSPa)-soluble protein fractions from glandless cottonseed. SDS-Gel electrophoresis separated CSPw and CSPa to 8 and 14 dominant polypeptide bands (110–10 kDa), respectively. Liquid chromatography-electrospray ionization-tandem mass spectrometry identified peptide fragments from 336 proteins. While the majority of peptides were identified as belonging to vicilin and legumin storage proteins, peptides from other functional and uncharacterized proteins were also detected. Based on the types (unique peptide count) and relative abundance (normalized total ion current) of the polypeptides detected by mass spectrometry, we found lower levels (abundance) and types of legumin isoforms, but higher levels and more fragments of vicilin-like antimicrobial peptides in glandless samples, compared to glanded samples. Differences in peptide fragment patterns of 2S albumin and oleosin were also observed between glandless and glanded protein samples. These differences might be due to the higher extraction recovery of proteins from glandless cottonseed as proteins from glanded cottonseed tend to be associated with gossypol, reducing extraction efficiency. This work enriches the fundamental knowledge of glandless cottonseed protein composition. For practical considerations, this peptide information will be helpful to allow better understanding of the functional and physicochemical properties of glandless cottonseed protein, and improving the potential for food or feed applications.

scholarly journals VectorMOD: Method for Bottom-Up Proteomic Characterization of rAAV Capsid Post-Translational Modifications and Vector Impurities

2021 ◽  
Vol 12 ◽  
Author(s):  
Neil G. Rumachik ◽  
Stacy A. Malaker ◽  
Nicole K. Paulk
Keyword(s):  
Mass Spectrometry ◽  
Process Development ◽  
Cell Protein ◽  
Macromolecular Complex ◽  
Bottom Up ◽  
Post Translational Modifications ◽  
Simple Step ◽  
Product And Process ◽  
High Doses ◽  
And Control

Progress in recombinant AAV gene therapy product and process development has advanced our understanding of the basic biology of this critical delivery vector. The discovery of rAAV capsid post-translational modifications (PTMs) has spurred interest in the field for detailed rAAV-specific methods for vector lot characterization by mass spectrometry given the unique challenges presented by this viral macromolecular complex. Recent concerns regarding immunogenic responses to systemically administered rAAV at high doses has highlighted the need for investigators to catalog and track potentially immunogenic vector lot components including capsid PTMs and PTMs on host cell protein impurities. Here we present a simple step-by-step guide for academic rAAV laboratories and Chemistry, Manufacturing and Control (CMC) groups in industry to perform an in-house or outsourced bottom-up mass spectrometry workflow to characterize capsid PTMs and process impurities.

scholarly journals Methodology to Quantify Collagen Subtypes and Crosslinks: Application in Minipig Cartilages

Cartilage ◽  
2021 ◽  
pp. 194760352110605
Author(s):  
Benjamin J. Bielajew ◽  
Jerry C. Hu ◽  
Kyriacos A. Athanasiou
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tissue Characterization ◽  
Collagen Type ◽  
Facet Joint ◽  
Type I ◽  
Collagen Types ◽  
Collagen Crosslinks ◽  
Bottom Up ◽  
Ms Analysis

Introduction This study develops assays to quantify collagen subtypes and crosslinks with liquid chromatography-mass spectrometry (LC-MS) and characterizes the cartilages in the Yucatan minipig. Methods For collagen subtyping, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed on tissues digested in trypsin. For collagen crosslinks, LC-MS analysis was performed on hydrolysates. Samples were also examined histologically and with bottom-up proteomics. Ten cartilages (femoral condyle, femoral head, facet joint, floating rib, true rib, auricular cartilage, annulus fibrosus, 2 meniscus locations, and temporomandibular joint disc) were analyzed. Results The collagen subtyping assay quantified collagen types I and II. The collagen crosslinks assay quantified mature and immature crosslinks. Collagen subtyping revealed that collagen type I predominates in fibrocartilages and collagen type II in hyaline cartilages, as expected. Elastic cartilage and fibrocartilages had more mature collagen crosslink profiles than hyaline cartilages. Bottom-up proteomics revealed a spectrum of ratios between collagen types I and II, and quantified 42 proteins, including 24 collagen alpha-chains and 12 minor collagen types. Discussion The novel assays developed in this work are sensitive, inexpensive, and use a low operator time relative to other collagen analysis methods. Unlike the current collagen assays, these assays quantify collagen subtypes and crosslinks without an antibody-based approach or lengthy chromatography. They apply to any collagenous tissue, with broad applications in tissue characterization and tissue engineering. For example, a novel finding of this work was the presence of a large quantity of collagen type III in the white-white knee meniscus and a spectrum of hyaline and fibrous cartilages.

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