scholarly journals Salt-Mediated Organic Solvent Precipitation for Enhanced Recovery of Peptides Generated by Pepsin Digestion

Proteomes ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 44
Author(s):  
Venus Baghalabadi ◽  
Habib Razmi ◽  
Alan Doucette
Keyword(s):  
Enhanced Recovery ◽  
Sequence Coverage ◽  
High Recovery ◽  
Pellet Fraction ◽  
Hydrophobic Peptides ◽  
Salt Ions ◽  
Low Mass ◽  
High Concentrations ◽  
Digestion Step ◽  
Protein Sequence Coverage

Conventional solvent-based precipitation makes it challenging to obtain a high recovery of low mass peptides. However, we previously demonstrated that the inclusion of salt ions, specifically ZnSO4, together with high concentrations of acetone, maximizes the recovery of peptides generated from trypsin digestion. We herein generalized this protocol to the rapid (5 min) precipitation of pepsin-digested peptides recovered from acidic matrices. The precipitation protocol extended to other organic solvents (acetonitrile), with high recovery from dilute peptide samples permitting preconcentration and purification. Mass spectrometry profiling of pepsin-generated peptides demonstrated that the protocol captured peptides as small as 800 u, although with a preferential bias towards recovering larger and more hydrophobic peptides. The precipitation protocol was applied to rapidly quench, concentrate, and purify pepsin-digested samples ahead of MS. Complex mixtures of yeast and plasma proteome extracts were successfully precipitated following digestion, with over 95% of MS-identified peptides observed in the pellet fraction. The full precipitation workflow—including the digestion step—can be completed in under 10 min, with direct MS analysis of the recovered peptide pellets showing exceptional protein sequence coverage.

scholarly journals Biocatalytic Transformation of 5-Hydroxymethylfurfural into 2,5-di(hydroxymethyl)furan by a Newly Isolated Fusarium striatum Strain

Catalysts ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 216
Author(s):  
Alberto Millán ◽  
Núria Sala ◽  
Mercè Torres ◽  
Ramon Canela-Garayoa
Keyword(s):  
Fusarium Species ◽  
Inoculum Size ◽  
High Recovery ◽  
23 Factorial Design ◽  
Organic Solvent Extraction ◽  
Working Volume ◽  
The Media ◽  
Substrate Feeding ◽  
High Concentrations ◽  
Reaction Media

The compound 2,5-di(hydroxymethyl)furan (DHMF) is a high-value chemical block that can be synthesized from 5-hydroxymethylfurfural (HMF), a platform chemical that results from the dehydration of biomass-derived carbohydrates. In this work, the HMF biotransformation capability of different Fusarium species was evaluated, and F. striatum was selected to produce DHMF. The effects of the inoculum size, glucose concentration and pH of the media over DHMF production were evaluated by a 23 factorial design. A substrate feeding approach was found suitable to overcome the toxicity effect of HMF towards the cells when added at high concentrations (>75 mM). The process was successfully scaled-up at bioreactor scale (1.3 L working volume) with excellent DHMF production yields (95%) and selectivity (98%). DHMF was purified from the reaction media with high recovery and purity by organic solvent extraction with ethyl acetate.

scholarly journals Increased Single-Spectrum Top-Down Protein Sequence Coverage in Trapping Mass Spectrometers with Chimeric Ion Loading

2020 ◽  
Vol 92 (18) ◽  
pp. 12193-12200
Author(s):  
Chad R. Weisbrod ◽  
Lissa C. Anderson ◽  
Joseph B. Greer ◽  
Caroline J. DeHart ◽  
Christopher L. Hendrickson
Keyword(s):  
Protein Sequence ◽  
Sequence Coverage ◽  
Top Down ◽  
Single Spectrum ◽  
Mass Spectrometers ◽  
Protein Sequence Coverage

scholarly journals A Colorimetric Method for Monitoring Tryptic Digestion Prior to Shotgun Proteomics

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Richard I. Somiari ◽  
Kutralanathan Renganathan ◽  
Stephen Russell ◽  
Steven Wolfe ◽  
Florentina Mayko ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Colorimetric Method ◽  
Shotgun Proteomics ◽  
Tryptic Digestion ◽  
Protein Digestion ◽  
Sequence Coverage ◽  
Time Sample ◽  
Expensive Equipment ◽  
Average Sequence ◽  
Digestion Step

Tryptic digestion is an important preanalytical step in shotgun proteomics because inadequate or excessive digestion can result in a failed or incomplete experiment. Unfortunately, this step is not routinely monitored before mass spectrometry because methods available for protein digestion monitoring either are time/sample consuming or require expensive equipment. To determine if a colorimetric method (ProDM Kit) can be used to identify the extent of tryptic digestion that yields the best proteomics outcome, plasma and serum digested for 8 h and 24 h were screened with ProDM, Bioanalyzer, and LC/MS/MS, and the effect of digestion on the number of proteins identified and sequence coverage was compared. About 6% and 16% less proteins were identified when >50% of proteins were digested in plasma and serum, respectively, compared to when ~46% of proteins were digested. Average sequence coverage for albumin, haptoglobin, and serotransferrin after 2 h, 8 h, and 24 h digestion was 52%, 45%, and 45% for serum and 54%, 47%, and 42% for plasma, respectively. This paper reiterates the importance of optimizing the tryptic digestion step and demonstrates the extent to which ProDM can be used to monitor and standardize protein digestion to achieve better proteomics outcomes.

scholarly journals Histidine decarboxylase from rat mast cells. Enhanced recovery in cell-free extracts and isotopic labelling

1988 ◽  
Vol 249 (1) ◽  
pp. 297-300 ◽  
Author(s):  
R S Feldberg ◽  
D A Iannitti ◽  
D E Cochrane
Keyword(s):  
Active Site ◽  
Gel Electrophoresis ◽  
Histidine Decarboxylase ◽  
Enhanced Recovery ◽  
Cell Free Extract ◽  
Isotopic Labelling ◽  
High Recovery ◽  
Gradient Gel Electrophoresis ◽  
Centrifugation Step ◽  
Rat Mast Cells

A procedure for obtaining rat mast-cell histidine decarboxylase in greater than 50% yield in cell-free extracts was developed. The enzyme was found in the supernatant fractions from a 3,500 g and a 105,000 g centrifugation step and was demonstrated to be sensitive to inhibition by alpha-fluoromethylhistidine but not by phenylalanine. Although the enzyme shows a half-life of only 3 h in cell-free extract, the initial high recovery of activity allowed for active-site labelling of the enzyme by [3H]histidine and NaBH4. Labelled protein migrated on non-denaturing polyacrylamide-gradient-gel electrophoresis as a 55,000 Da species.

scholarly journals Effects of Salts on Structural, Physicochemical and Rheological Properties of Low-Methoxyl Pectin/Sodium Caseinate Complex

Foods ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2009
Author(s):  
Shengyu Fan ◽  
Fang Fang ◽  
Ailing Lei ◽  
Jiong Zheng ◽  
Fusheng Zhang
Keyword(s):  
Complex System ◽  
Particle Size ◽  
Secondary Structure ◽  
Zeta Potential ◽  
Rheological Properties ◽  
Food Industry ◽  
Protein Complexes ◽  
Sodium Caseinate ◽  
Salt Ions ◽  
High Concentrations

The addition of salts is an effective way to improve the properties of polysaccharide/protein complexes for use in foods. However, there is no comparative study on the effects of different ions on the complex system of low methoxyl pectin (LMP)/ sodium caseinate (CAS) complex. The effects of different concentrations of three salt ions (Na+, K+, Ca2+) on the physicochemical and rheological properties of the LMP/CAS complex were determined in this study, and the structure of LMP/CAS complex was characterized. The results showed that the addition of these three salt ions affected zeta potential, particle size, and turbidity of the LMP/CAS complex, and lead the LMP/CAS complex to form a more regular and uniform network structure, which helped improve its stability, solubility, and rheological properties. The particle size and turbidity value of the complex achieved with Ca2+ were higher than those obtained using Na+ and K+. Moreover, the secondary structure of the proteins in the complex changed to adding high concentrations of Ca2+. Our study provides valuable information for the application of the LMP/CAS complex in the food industry.

scholarly journals Biocatalytic Transformation of 5-Hydroxymethylfurfural into 2,5-Di(hydroxymethyl)furan by a Newly Isolated Fusarium striatum Strain

2021 ◽  
Author(s):  
Alberto Millán ◽  
Núria Sala ◽  
Mercè Torres ◽  
Ramon Canela-Garayoa
Keyword(s):  
Fusarium Species ◽  
Inoculum Size ◽  
High Recovery ◽  
23 Factorial Design ◽  
Toxicity Effect ◽  
Organic Solvent Extraction ◽  
The Media ◽  
Substrate Feeding ◽  
High Concentrations ◽  
Reaction Media

2,5-Di(hydroxymethyl)furan (DHMF) is a high-value chemical block than can be synthesized from 5-hydroxymethylfurfural (HMF), a platform chemical that results from the dehydration of biomass-derived carbohydrates. In this work, the HMF biotransformation capability of different Fusarium species was evaluated and F. striatum was selected to produce DHMF. The effects of the inoculum size, glucose concentration and pH of the media over DHMF production were evalu-ated by a 23 factorial design. A substrate feeding approach was found suitable to overcome the toxicity effect of HMF towards the cells when added at high concentrations (>75 mM). The pro-cess was successfully scaled-up at bioreactor scale (1.3 L) with excellent DHMF production yields (95%) and selectivities (98%). DHMF was purified from the reaction media with high recovery and purity by organic solvent extraction with ethyl acetate.

Application of electro-Fenton oxidation for the detoxification of olive mill wastewater phenolic compounds

2004 ◽  
Vol 49 (4) ◽  
pp. 97-102 ◽  
Author(s):  
S. Khoufi ◽  
H. Aouissaoui ◽  
M. Penninckx ◽  
S. Sayadi
Keyword(s):  
Phenolic Compounds ◽  
Olive Mill Wastewater ◽  
Fenton Oxidation ◽  
Efficient Procedure ◽  
Olive Mill Wastewaters ◽  
Molecular Masses ◽  
Low Mass ◽  
Pre Treatment ◽  
High Concentrations ◽  
Olive Mill

Olive mill wastewaters are powerful pollutants that are difficult to treat as they contain high concentrations of recalcitrant and toxic polyphenolics of different molecular masses. We report in this paper that pre-treatment of phenolic fractions extracted from olive mill wastewaters as well as a synthetic phenolic mixture by the electro-Fenton method is a highly efficient procedure in which low mass phenolics are polymerised to polyphenolics and removed by precipitation. This decreases the toxicity load by 78% and makes the waste amenable to further anaerobic post-treatment.

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