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 Vibratory Reaction Unit for the Rapid Analysis of Proteins and Glycochains

2007 ◽  
Vol 2 ◽  
pp. 117739010700200 ◽  
Author(s):  
Yukie Sasakura ◽  
Makoto Nogami ◽  
Noriko Kobayashi ◽  
Katsuhiro Kanda
Keyword(s):  
Mass Spectrometry ◽  
Solid Surface ◽  
Protein Identification ◽  
Immobilized Enzymes ◽  
Rapid Analysis ◽  
Single Step ◽  
Protein Digestion ◽  
Sequence Coverage ◽  
Micro Scale ◽  
Reaction Unit

A protein digestion system using immobilized enzymes for protein identification and glycochain analyses has been developed, and a vibration reaction unit for micro-scale sample convection on an enzyme-immobilized solid surface was constructed. BSA as a model substrate was digested by this unit, and was successfully identified by mass spectrometry (MS) analyses. Compared to the conventional liquid-phase digestion, the reaction unit increased the number of matched peptides from 9 to 26, protein score from 455 to 1247, and sequence coverage from 21% to 48%. Glycopeptidase F (NGF), an enzyme that cleaves N-glycans from glycoproteins, was also immobilized and used to remove the glycochains from human immunoglobulin G (IgG). Trypsin and NGF were immobilized on the same solid surface and used to remove glycochains from IgG in single-step. Glycochains were labeled with fluorescent reagent and analyzed by HPLC. Several peaks corresponding to the glycochains of IgG were detected. These results suggested that the single-step digestion system, by immobilized multiple enzymes (trypsin and NGF) would be effective for the rapid structural analysis of glycoproteins.

200. The colorimetric determination of pH in milk and whey by means of the “Wulff” pH tester

1938 ◽  
Vol 9 (3) ◽  
pp. 336-338 ◽  
Author(s):  
E. Aschaffenburg
Keyword(s):  
Colorimetric Method ◽  
Cheddar Cheese ◽  
Titratable Acidity ◽  
Colorimetric Determination ◽  
Expensive Equipment ◽  
Hydrogenion Concentration ◽  
The Relationship

It has been repeatedly pointed out(1, 2, 3) that the properties of cheese during the different stages of its manufacture should be correlated with the hydrogenion concentration rather than with the titratable acidity. Little systematic work has, however, so far been carried out in this direction, except for a study of the relationship between pH and titratable acidity in Cheddar cheese by Brown & Price(4). In planning work on similar lines, it was realized that the potentiometric methods of determining pH require expensive equipment and skilled attention, so that a supplementary colorimetric method, if sufficiently accurate to indicate the major changes in pH, should appeal more strongly to the practical cheesemaker on account of its cheapness and simplicity and the ease with which the outfit can be transported.

Determination and Identification of Daminozide in Fresh Fruits Purchased in Japan

1990 ◽  
Vol 53 (8) ◽  
pp. 689-692
Author(s):  
YUMIKO NAKAMURA ◽  
YUKARI HASEGAWA ◽  
YASUHIDE TONOGAI ◽  
YOSHIO ITO
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Alkaline Solution ◽  
Colorimetric Method ◽  
Gas Chromatography Mass Spectrometry ◽  
Unsymmetrical Dimethylhydrazine ◽  
Chromatography Mass Spectrometry ◽  
Trade Name ◽  
Red Color

The concentration of residual daminozide (trade name of formulation: Alar or B-Nine) in cherries, grapes, peaches, and apples purchased in Japan was investigated using the colorimetric method. Daminozide hydrolyzes in boiling strong alkaline solution to release unsymmetrical dimethylhydrazine which is distilled and reacted with trisodium pentacyanoamine ferroate to form a specific red color at pH 4.5. This color is measured spectrophotometrically. The levels of daminozide detected were 0.07–1.39 ppm in cherries (11 samples), 0–0.36 ppm in grapes (10 samples), 0–0.42 ppm in peaches (9 samples), and 0–0.88 ppm in apples (10 samples). Furthermore, daminozide in each sample was identified as methyl daminozide by the gas chromatography/mass spectrometry (GC/MS) method.

scholarly journals New insights into Trypanosoma cruzi evolution and genotyping based on system-wide protein expression profiles (PhyloQuant)

2020 ◽  
Author(s):  
Simon Ngao Mule ◽  
Andrè Guillherme da Costa Martins ◽  
Livia Rosa-Fernandes ◽  
Gilberto Santos de Oliveira ◽  
Carla Monadeli Rodrigues ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Trypanosoma Cruzi ◽  
Protein Expression ◽  
Large Scale ◽  
Expression Profiles ◽  
Shotgun Proteomics ◽  
Close Correlation ◽  
Evolutionary Relationships ◽  
Trypanosome Species ◽  
Protein Expression Profiles

AbstractThe etiological agent of Chagas disease, Trypanosoma cruzi, is subdivided into seven genetic subdivisions termed discrete typing units (DTUs), TcI-TcVI and Tcbat. The relevance of T. cruzi genetic diversity to the variable clinical course of the disease, virulence, pathogenicity, drug resistance, transmission cycles and ecological distribution justifies the concerted efforts towards understanding the population structure of T. cruzi strains. In this study, we introduce a novel approach termed ‘phyloquant’ to infer the evolutionary relationships and assignment of T. cruzi strains to their DTUs based on differential protein expression profiles evidenced by bottom up large scale mass spectrometry-based quantitative proteomic features. Mass spectrometry features analyzed using parsimony (MS1, iBAQ and LFQ) showed a close correlation between protein expression and T. cruzi DTUs and closely related trypanosome species. Although alternative topologies with minor differences between the three MS features analyzed were demonstrated, we show congruence to well accepted evolutionary relationships of T. cruzi DTUs; in all analyses TcI and Tcbat were sister groups, and the parental nature of genotype TcII and the hybrid genotypes TcV/TcVI were corroborated. Character mapping of genetic distance matrices based on phylogenetics and phyloquant clustering showed statistically significant correlations. We propose the first quantitative shotgun proteomics approach as a complement strategy to the genetic-based assignment of T. cruzi strains to DTUs and evolutionary inferences. Moreover, this approach allows for the identification of differentially regulated and strain/DTU/species-specific proteins, with potential application in the identification of strain/DTU specific biomarkers and candidate therapeutic targets. In addition, the correlation between multi-gene protein expression and divergence of trypanosome species was evaluated, adding another level to understand the genetic subdivisions among T. cruzi DTUs.

Abstract P513: Adenylosuccinate Synthase Is A Novel Methylation Target Of Smyd1

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Magnus Creed ◽  
Marta Szulik ◽  
Ryan Bia ◽  
Chris Tracy ◽  
Aman Makaju ◽  
...  
Keyword(s):  
Heart Failure ◽  
Mass Spectrometry ◽  
Uric Acid ◽  
Purine Metabolism ◽  
Metabolic Enzyme ◽  
Mouse Heart ◽  
Lysine Methylation ◽  
Sequence Coverage ◽  
Underlying Mechanisms

Among the metabolic shifts in chronic heart failure is a dysregulation of purine metabolism, which has been shown to negatively impact patient outcomes, especially in individuals affected by hypertension, diabetes, and congestive heart failure, via increased serum uric acid levels and cellular oxidative stress. The underlying mechanisms which drive these changes in purine metabolism in the cardiomyocyte and ultimately reactive oxygen species and uric acid accumulation in heart failure patients remain largely unknown. We recently discovered that the methyltransferase Smyd1 interacts with the metabolic enzyme Adss (Adenylosuccinate synthetase), a key component of purine metabolism in the heart involved in AMP synthesis, via co-immunoprecipitation. We confirmed this novel interaction between Smyd1b and Adss in mouse heart and cultured primary cardiomyocytes, which is further enhanced during phenylephrine-induced hypertrophy in the latter. Our hypothesis was that Smyd1b methylates Adss to regulate its activity, therefore, we examined lysine methylation on Adss via western blotting and mass spectrometry and quantified its ability to convert IMP to sAMP in vitro in the presence and absence of Smyd1b. Using a pan-methylation antibody we initially detected di- and tri-methylation on Adss which was increased in the presence of Smyd1b. Then utilizing bottom-up proteomics, we achieved 98% sequence coverage of Adss via mass spectrometry and identified trimethylation on K373 only in the presence of Smyd1b. In addition, utilizing an enzymatic assay in vitro we have shown that Smyd1b enhances the activity of Adss as it converts IMP to s-AMP. While it has been well-established that the activities of metabolic enzymes are modulated via post-translational modifications (e.g. phosphorylation, acetylation), we believe this is the first report of a metabolic enzyme regulated by lysine methylation. These exciting results highlight a novel role for Smyd1b in regulating purine metabolism in the myocyte and begin to lay the groundwork for examining this mechanism in the setting of disease.

Tryptic digestion of human serum for proteomic mass spectrometry automated by centrifugal microfluidics

Lab on a Chip ◽  
2020 ◽  
Vol 20 (16) ◽  
pp. 2937-2946
Author(s):  
J.-N. Klatt ◽  
M. Depke ◽  
N. Goswami ◽  
N. Paust ◽  
R. Zengerle ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Human Serum ◽  
Centrifugal Microfluidics ◽  
Tryptic Digestion

Tryptic digestion of human serum automated by centrifugal microfluidics.

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