Journal ArticleDOI
Acetone precipitation of proteins and the modification of peptides.
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TLDR
It is shown that a trace amount of residual acetone in the precipitated protein, can, after proteolysis, lead to selective modification of peptides predominantly those in which a glycine residue is the second amino acid, probably generating a relatively stable derivative that, under gas phase conditions, generates a y(1) ion of the same mass as proline.Abstract:
Acetone precipitation is a common method for precipitation and concentration of proteins. We show here that a trace amount of residual acetone in the precipitated protein, can, after proteolysis, lead to selective modification of peptides predominantly those in which a glycine residue is the second amino acid, probably generating a relatively stable derivative that, under gas phase conditions, generates a y(1) ion of the same mass as proline. This modification is detectable by either MALDI-ToF or ESI-ion trap mass spectrometry and under normal sample preparation conditions is incomplete. The derivatization occurs in the condensed phase and is sufficiently stable that the modified peptide can elute on reversed phase chromatography at a different time to the unmodified peptide. Acetone precipitation is such a commonly used procedure in protein sample preparation for proteomics that some caution may be warranted. A significant number of peptides (about 5% of a typical proteome) meet the requirements for this reaction and could, therefore, change the outcome of studies.read more
Citations
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Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis.
Ewelina Fic,Sylwia Kedracka-Krok,Urszula Jankowska,Artur Pirog,Marta Dziedzicka-Wasylewska,Marta Dziedzicka-Wasylewska +5 more
TL;DR: It was found that for four of the rat brain structures, precipitation with chloroform/methanol and acetone delivered the highest protein recovery for top‐down proteomic analysis; however, TCA precipitation resulted in good protein separation and the highest number of protein spots in 2‐DE.
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Proteomic analysis of exosomal cargo: the challenge of high purity vesicle isolation
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Biomarker discovery in mass spectrometry-based urinary proteomics
TL;DR: Promising technologies and strategies in the MS‐based biomarker discovery process are highlighted, including study design, sample preparation, protein quantification, instrumental platforms, and bioinformatics.
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TL;DR: Conclusively, both label-free and iTRAQ (when combined to peptide fractionation) provide high proteome coverage and apparently valid predictions in terms of differential expression, neverthelesslabel-free provides higher sequence coverage and ultimately detects a higher number of differentially expressed proteins.
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Bimetallic cerium/copper organic framework-derived cerium and copper oxides embedded by mesoporous carbon: Label-free aptasensor for ultrasensitive tobramycin detection.
Shijun Wang,Zhenzhen Li,Fenghe Duan,Bin Hu,Linghao He,Minghua Wang,Nan Zhou,Qiaojuan Jia,Zhihong Zhang +8 more
TL;DR: The proposed aptasensing approach based on bimetallic CeO2/CuOx@mC has a considerable potential for the quantitative detection of antibiotics in the food safety and biomedical field and demonstrates that the proposed apt asensor is substantially superior to those previously reported in the literature.
References
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Journal ArticleDOI
Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation.
Terrence L. Geiger,Steven Clarke +1 more
TL;DR: These studies indicate that both aspartic acid and asparagine residues may be hot spots for the nonenzymatic degradation of proteins, especially in cells such as erythrocytes and eye lens, where these macromolecules must function for periods of about 120 days and 80 years, respectively.
Journal ArticleDOI
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Jeffrey C. Silva,Richard Denny,Craig A. Dorschel,Marc V. Gorenstein,Ignatius J. Kass,Guo-Zhong Li,Therese McKenna,Michael J. Nold,Keith Richardson,Phillip Young,Scott J. Geromanos +10 more
TL;DR: The principal focus of this paper will demonstrate the quantitative aspects of the methodology and continue with a discussion of the associated, complementary qualitative capabilities.
Journal ArticleDOI
Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides
TL;DR: The successful design and construction of an artificial gene encoding a concatenation of tryptic peptides (QCAT protein) from several chick skeletal muscle proteins and features for quantification and purification are reported.
Journal ArticleDOI
Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes
Julie M. Pratt,Deborah M. Simpson,Mary K. Doherty,Jenny Rivers,Simon J. Gaskell,Robert J. Beynon +5 more
TL;DR: This protocol details the methods for the design, expression, labeling, purification, characterization and use of the QconCATs in the absolute quantification of complex protein mixtures.
Journal ArticleDOI
Simultaneous Qualitative and Quantitative Analysis of theEscherichia coli Proteome A Sweet Tale
Jeffrey C. Silva,Richard Denny,Craig A. Dorschel,Marc V. Gorenstein,Guo-Zhong Li,Keith Richardson,Daniel Wall,Scott J. Geromanos +7 more
TL;DR: In this paper, a label-free, LCMS acquisition method observes all detectable, eluting peptides and their corresponding fragment ions, and the change in relative abundance of the corresponding proteins was measured from peptides common to both conditions.