scholarly journals Bone protein “extractomics”: comparing the efficiency of bone protein extractions ofGallus gallusin tandem mass spectrometry, with an eye towards paleoproteomics

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2603 ◽  
Author(s):  
Elena R. Schroeter ◽  
Caroline J. DeHart ◽  
Mary H. Schweitzer ◽  
Paul M. Thomas ◽  
Neil L. Kelleher
Keyword(s):  
Mass Spectrometry ◽  
Protein Extraction ◽  
Bone Matrix ◽  
Extraction Methods ◽  
Protein Recovery ◽  
Sequence Coverage ◽  
Future Directions ◽  
Critical Data ◽  
Bone Proteins ◽  
Extraction Buffer

Proteomic studies of bone require specialized extraction protocols to demineralize and solubilize proteins from within the bone matrix. Although various protocols exist for bone protein recovery, little is known about how discrete steps in each protocol affect the subset of the bone proteome recovered by mass spectrometry (MS) analyses. Characterizing these different “extractomes” will provide critical data for development of novel and more efficient protein extraction methodologies for fossils. Here, we analyze 22 unique sub-extractions of chicken bone and directly compare individual extraction components for their total protein yield and diversity and coverage of bone proteins identified by MS. We extracted proteins using different combinations and ratios of demineralizing reagents, protein-solubilizing reagents, and post-extraction buffer removal methods, then evaluated tryptic digests from 20 µg aliquots of each fraction by tandem MS/MS on a 12T FT-ICR mass spectrometer. We compared total numbers of peptide spectral matches, peptides, and proteins identified from each fraction, the redundancy of protein identifications between discrete steps of extraction methods, and the sequence coverage obtained for select, abundant proteins. Although both alpha chains of collagen I (the most abundant protein in bone) were found in all fractions, other collagenous and non-collagenous proteins (e.g., apolipoprotein, osteonectin, hemoglobin) were differentially identified. We found that when a standardized amount of extracted proteins was analyzed, extraction steps that yielded the most protein (by weight) from bone were oftennotthe ones that produced the greatest diversity of bone proteins, or the highest degree of protein coverage. Generally, the highest degrees of diversity and coverage were obtained from demineralization fractions, and the proteins found in the subsequent solubilization fractions were highly redundant with those in the previous fraction. Based on these data, we identify future directions and parameters to consider (e.g., proteins targeted, amount of sample required) when applying discrete parts of these protocols to fossils.

scholarly journals Review: current trends in oat protein recovery and utilization in aqueous food systems

2021 ◽  
Author(s):  
Darius Sargautis ◽  
◽  
Tatjana Kince ◽  
Vanda Sargautiene ◽  
Keyword(s):  
Functional Properties ◽  
Food Systems ◽  
Protein Extraction ◽  
Protein Solubility ◽  
Extraction Methods ◽  
Scientific Data ◽  
Protein Recovery ◽  
Protein Isolation ◽  
Dry Fractionation ◽  
Isolation Methods

Oat protein itself, as a substance, has extensively been studied providing information on its nutritional value, some functional properties and possible applicability in food systems. Chosen protein isolation methods and technological aspects define final composition of obtained oat protein product, its concentration, nutrition value and its functionality in food industry. Scientific data on oat protein recovery methods, typically relying on protein solubility or dry fractionation, provides an insufficient knowledge about the success in commercialization of oat protein recovery technologies and their derivatives in form of oat protein. The aim of the study was to analyse and summarize the research findings on oat protein extraction methods and functional properties of oat protein. Semi-systematic, monographic methods were used to analyse the oat protein isolation techniques, functional properties of oat protein in aqueous food systems, covering the latest information on oat protein extraction methods. Wet and dry isolation methods were demonstrated as main methods in oat protein extraction. Functional properties of oat protein, such as thermal stability, solubility, emulsification, water hydration capacity and foaming were reviewed and evaluated, identifying limitations and protein alterations which occur through the oat protein extraction process. The study provides recent trends in oat protein recovery technologies, along with an overview of current and potential oat protein utilization in food systems.

scholarly journals Proteomic method to extract, concentrate, digest and enrich peptides from fossils with coloured (humic) substances for mass spectrometry analyses

2019 ◽  
Vol 6 (8) ◽  
pp. 181433 ◽  
Author(s):  
Elena R. Schroeter ◽  
Kevin Blackburn ◽  
Michael B. Goshe ◽  
Mary H. Schweitzer
Keyword(s):  
Mass Spectrometry ◽  
Organic Matter ◽  
Sample Preparation ◽  
Humic Substances ◽  
Protein Loss ◽  
Sequence Coverage ◽  
Centrifugal Stage ◽  
Recent Advances ◽  
Extraction Buffer ◽  
Multiple Samples

Humic substances are breakdown products of decaying organic matter that co-extract with proteins from fossils. These substances are difficult to separate from proteins in solution and interfere with analyses of fossil proteomes. We introduce a method combining multiple recent advances in extraction protocols to both concentrate proteins from fossil specimens with high humic content and remove humics, producing clean samples easily analysed by mass spectrometry (MS). This method includes: (i) a non-demineralizing extraction buffer that eliminates protein loss during the demineralization step in routine methods; (ii) filter-aided sample preparation (FASP) of peptides, which concentrates and digests extracts in one filter, allowing the separation of large humics after digestion; (iii) centrifugal stage tipping, which further clarifies and concentrates samples in a uniform process performed simultaneously on multiple samples. We apply this method to a moa fossil (approx. 800–1000 years) dark with humic content, generating colourless samples and enabling the detection of more proteins with greater sequence coverage than previous MS analyses on this same specimen. This workflow allows analyses of low-abundance proteins in fossils containing humics and thus may widen the range of extinct organisms and regions of their proteomes we can explore with MS.

scholarly journals Biorefinery of Rubber Seed Kernels for Comprehensive Utilisation

2020 ◽  
Author(s):  
Miao Yang ◽  
Wenlei Zhu ◽  
Hui Cao
Keyword(s):  
Oil Recovery ◽  
Protein Extraction ◽  
Protein Denaturation ◽  
Extraction Methods ◽  
Alkaline Treatment ◽  
Protein Recovery ◽  
Recovery Efficiency ◽  
Rubber Seed ◽  
Rubber Production ◽  
Comprehensive Utilization

Abstract Rubber seeds are a by-product of rubber production that is rich in oil and protein. Upgrading of rubber seeds to produce proteins, oils and feedstock can generate additional revenue for rubber production and reduce waste. The present study investigates the effects of different pre-treatments and extraction methods to determine the optimal methods to produce oil and protein from rubber seed kernels. Mechanical expulsion using a screw press and solvent extraction using n-hexane were employed for oil separation. The highest oil recovery efficiency of 95% was obtained using rubber seed meal that was pre-dried at 105 ℃. The sequential water-alkaline treatment was ideal for achieving high protein recovery while reducing the protein denaturation that can result from high operating temperatures and organic solvent contact. Over 90% of the total protein from rubber seed kernels could be recovered. Separating oil from kernels using hexane followed by protein extraction from the meals by enzymatic treatment provides a suitable method for comprehensive utilization of rubber seeds.

Compatibility of plant protein extraction methods with mass spectrometry for proteome analysis

Plant Science ◽  
2009 ◽  
Vol 176 (1) ◽  
pp. 99-104 ◽  
Author(s):  
Inder S. Sheoran ◽  
Andrew R.S. Ross ◽  
Douglas J.H. Olson ◽  
Vipen K. Sawhney
Keyword(s):  
Mass Spectrometry ◽  
Protein Extraction ◽  
Proteome Analysis ◽  
Extraction Methods ◽  
Plant Protein

scholarly journals Optimisation of Protein Extraction from Medicinal Cannabis Mature Buds for Bottom-Up Proteomics

Molecules ◽  
2019 ◽  
Vol 24 (4) ◽  
pp. 659 ◽  
Author(s):  
Delphine Vincent ◽  
Simone Rochfort ◽  
German Spangenberg
Keyword(s):  
Mass Spectrometry ◽  
Cannabis Sativa ◽  
Protein Extraction ◽  
Guanidine Hydrochloride ◽  
Shotgun Proteomics ◽  
Extraction Methods ◽  
Reproductive Organs ◽  
Apical Buds ◽  
Liquid Chromatography Tandem Mass ◽  
Medicinal Cannabis

Medicinal cannabis is used to relieve the symptoms of certain medical conditions, such as epilepsy. Cannabis is a controlled substance and until recently was illegal in many jurisdictions. Consequently, the study of this plant has been restricted. Proteomics studies on Cannabis sativa reported so far have been primarily based on plant organs and tissues other than buds, such as roots, hypocotyl, leaves, hempseeds and flour. As far as we know, no optimisation of protein extraction from cannabis reproductive tissues has been attempted. Therefore, we set out to assess different protein extraction methods followed by mass spectrometry-based proteomics to recover, separate and identify the proteins of the reproductive organs of medicinal cannabis, apical buds and isolated trichomes. Database search following shotgun proteomics was limited to protein sequences from C. sativa and closely related species available from UniprotKB. Our results demonstrate that a buffer containing the chaotrope reagent guanidine hydrochloride recovers many more proteins than a urea-based buffer. In combination with a precipitation with trichloroacetic acid, such buffer proved optimum to identify proteins using a trypsin digestion followed by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) analyses. This is validated by focusing on enzymes involved in the phytocannabinoid pathway.

scholarly journals Assessment and refinement of sample preparation methods for deep and quantitative plant proteome profiling

2018 ◽  
Author(s):  
Gaoyuan Song ◽  
Polly Y Yingshan Hsu ◽  
Justin W. Walley
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Extraction ◽  
Leaf Tissue ◽  
Extraction Methods ◽  
Label Free ◽  
Preparation Methods ◽  
Proteome Profiling ◽  
Leaf Tissues ◽  
Sample Preparation Methods

SummaryA major challenge in the field of proteomics is obtaining high quality peptides for comprehensive proteome profiling by liquid chromatography mass spectrometry for many organisms. Here we evaluate and modify a range of sample preparation methods using photosynthetically active Arabidopsis leaf tissues from several developmental timepoints. We find that inclusion of FASP-based on filter digestion improves all protein extraction methods tested. Ultimately, we show that a detergent-free urea-FASP approach enables deep and robust quantification of leaf proteomes. For example, from 4-day-old leaf tissue we profiled up to 11,690 proteins from a single sample replicate. This method should be broadly applicable to researchers working on difficult to process samples from a range of plant and non-plant organisms.AbbreviationsChloroMethanol/Chloroform ExtractionFASPFilter Aided Sample PrepGOGene OntologyIAAIodoacetamideLFQLabel Free QuantificationMS/MSTandem mass spectrometryTFTranscription FactorUAUrea Extraction1D1 Dimensional2D2 Dimensional

scholarly journals Comparative analysis of protein extraction methods for the identification of seafood-borne pathogenic and spoilage bacteria by MALDI-TOF mass spectrometry

2010 ◽  
Vol 2 (12) ◽  
pp. 1941 ◽  
Author(s):  
Karola Böhme ◽  
Inmaculada C. Fernández-No ◽  
Jorge Barros-Velázquez ◽  
Jose M. Gallardo ◽  
Benito Cañas ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Comparative Analysis ◽  
Protein Extraction ◽  
Extraction Methods ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof ◽  
Spoilage Bacteria

scholarly journals Biorefinery methods for extraction of oil and protein from rubber seed

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Miao Yang ◽  
Wenlei Zhu ◽  
Hui Cao
Keyword(s):  
Oil Recovery ◽  
Protein Extraction ◽  
Protein Denaturation ◽  
Extraction Methods ◽  
Alkaline Treatment ◽  
Protein Recovery ◽  
Recovery Efficiency ◽  
Rubber Seed ◽  
Rubber Production ◽  
Comprehensive Utilization

AbstractRubber seeds are a by-product of rubber production and are rich in oil and protein. Upgrading of rubber seeds to produce proteins, oils and feedstock can generate additional revenue for rubber production and reduce waste. The present study investigates the effects of different pre-treatments and extraction methods to determine the optimal methods to produce oil and protein from rubber seed kernels. Mechanical expulsion using a screw press and solvent extraction using n-hexane were employed for oil separation. The highest oil recovery efficiency of 95.12% was obtained using rubber seed meal that was pre-dried at 105 ℃. The sequential water–alkaline treatment was ideal for achieving high protein recovery while reducing the protein denaturation that can result from high operating temperatures and organic solvent contact. Over 90% of the total protein from rubber seed kernels could be recovered. Separating oil from kernels using hexane followed by protein extraction from the meals by enzymatic treatment provides a suitable method for comprehensive utilization of rubber seeds.

Development of a C12Im-Cl-assisted method for rapid sample preparation in proteomic application

2021 ◽  
Author(s):  
Chang Liu ◽  
Xiaoxia Si ◽  
Shumei Yan ◽  
Xinyuan Zhao ◽  
Xiaohong Qian ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Extraction ◽  
Sample Processing ◽  
Processing Methods ◽  
Proteomic Analyses

Chromatography and mass spectrometry (MS) techniques have greatly improved the power of proteomic analyses. However, sample processing methods, including protein extraction and digestion, before MS remain as bottlenecks in the...

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