A method for the extraction of the endogenous tryptic peptides (peptidome) from human EDTA plasma

2018 ◽  
Vol 549 ◽  
pp. 188-196 ◽  
Author(s):  
Jaimie Dufresne ◽  
Angelique Florentinus-Mefailoski ◽  
Pete Bowden ◽  
John G. Marshall
Keyword(s):  
Tryptic Peptides ◽  
Edta Plasma

scholarly journals Random and independent sampling of endogenous tryptic peptides from normal human EDTA plasma by liquid chromatography micro electrospray ionization and tandem mass spectrometry

2021 ◽  
Author(s):  
Jaimie Dufresne ◽  
Angelique Florentinus-Mefailoski ◽  
Juliet Ajambo ◽  
Ammara Ferwa ◽  
Peter Bowden ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Electrospray Ionization ◽  
Room Temperature ◽  
Tandem Mass ◽  
Plasma Samples ◽  
Tryptic Peptides ◽  
Edta Plasma ◽  
Normal Human

Background Normal human EDTA plasma samples were collected on ice, processed ice cold, and stored in a freezer at – 80 °C prior to experiments. Plasma test samples from the – 80 °C freezer were thawed on ice or intentionally warmed to room temperature. Methods Protein content was measured by CBBR binding and the release of alcohol soluble amines by the Cd ninhydrin assay. Plasma peptides released over time were collected over C18 for random and independent sampling by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) and correlated with X!TANDEM. Results Fully tryptic peptides by X!TANDEM returned a similar set of proteins, but was more computationally efficient, than “no enzyme” correlations. Plasma samples maintained on ice, or ice with a cocktail of protease inhibitors, showed lower background amounts of plasma peptides compared to samples incubated at room temperature. Regression analysis indicated that warming plasma to room temperature, versus ice cold, resulted in a ~ twofold increase in the frequency of peptide identification over hours–days of incubation at room temperature. The type I error rate of the protein identification from the X!TANDEM algorithm combined was estimated to be low compared to a null model of computer generated random MS/MS spectra. Conclusion The peptides of human plasma were identified and quantified with low error rates by random and independent sampling that revealed 1000s of peptides from hundreds of human plasma proteins from endogenous tryptic peptides.

scholarly journals Random and independent sampling of endogenous tryptic peptides from normal human EDTA plasma by liquid chromatography micro electrospray ionization and tandem mass spectrometry

2021 ◽  
Author(s):  
Jaimie Dufresne ◽  
Angelique Florentinus-Mefailoski ◽  
Juliet Ajambo ◽  
Ammara Ferwa ◽  
Peter Bowden ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Electrospray Ionization ◽  
Room Temperature ◽  
Tandem Mass ◽  
Plasma Samples ◽  
Tryptic Peptides ◽  
Edta Plasma ◽  
Normal Human

Background Normal human EDTA plasma samples were collected on ice, processed ice cold, and stored in a freezer at – 80 °C prior to experiments. Plasma test samples from the – 80 °C freezer were thawed on ice or intentionally warmed to room temperature. Methods Protein content was measured by CBBR binding and the release of alcohol soluble amines by the Cd ninhydrin assay. Plasma peptides released over time were collected over C18 for random and independent sampling by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) and correlated with X!TANDEM. Results Fully tryptic peptides by X!TANDEM returned a similar set of proteins, but was more computationally efficient, than “no enzyme” correlations. Plasma samples maintained on ice, or ice with a cocktail of protease inhibitors, showed lower background amounts of plasma peptides compared to samples incubated at room temperature. Regression analysis indicated that warming plasma to room temperature, versus ice cold, resulted in a ~ twofold increase in the frequency of peptide identification over hours–days of incubation at room temperature. The type I error rate of the protein identification from the X!TANDEM algorithm combined was estimated to be low compared to a null model of computer generated random MS/MS spectra. Conclusion The peptides of human plasma were identified and quantified with low error rates by random and independent sampling that revealed 1000s of peptides from hundreds of human plasma proteins from endogenous tryptic peptides.

scholarly journals Plasma Peptidome

2021 ◽  
Author(s):  
Jaimie Dufresne ◽  
Pete Bowden ◽  
Thanusi Thavarajah ◽  
Angelique Florentinus-Mefailoski ◽  
Zhuo Zhen Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ex Vivo ◽  
Sql Server ◽  
Plasma Samples ◽  
Tryptic Peptides ◽  
Esi Ms ◽  
Human Proteins ◽  
Edta Plasma ◽  
Best Fit ◽  
Gene Symbols

Background It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC–ESI–MS/MS to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations. Methods A systematic method for the organic fractionation of plasma peptides was applied to identify and quantify the endogenous tryptic peptides from human plasma from multiple institutions by C18 HPLC followed nano electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a linear quadrupole ion trap. The endogenous tryptic peptides, or tryptic phospho peptides (i.e. without exogenous digestion), were extracted in a mixture of organic solvent and water, dried and collected by preparative C18. The tryptic peptides from 6 institutions with 12 different disease and normal EDTA plasma populations, alongside ice cold controls for pre-analytical variation, were characterized by mass spectrometry. Each patient plasma was precipitated in 90% acetonitrile and the endogenous tryptic peptides extracted by a stepwise gradient of increasing water and then formic acid resulting in 10 sub-fractions. The fractionated peptides were manually collected over preparative C18 and injected for 1508 LC–ESI–MS/MS experiments analyzed in SQL Server R. Results Peptides that were cleaved in human plasma by a tryptic activity ex vivo provided convenient and sensitive access to most human proteins in plasma that show differences in the frequency or intensity of proteins observed across populations that may have clinical significance. Combination of step wise organic extraction of 200 μL of plasma with nano electrospray resulted in the confident identification and quantification ~ 14,000 gene symbols by X!TANDEM that is the largest number of blood proteins identified to date and shows that you can monitor the ex vivo proteolysis of most human proteins, including interleukins, from blood. A total of 15,968,550 MS/MS spectra ≥ E4 intensity counts were correlated by the SEQUEST and X!TANDEM algorithms to a federated library of 157,478 protein sequences that were filtered for best charge state (2+ or 3+) and peptide sequence in SQL Server resulting in 1,916,672 distinct best-fit peptide correlations for analysis with the R statistical system. SEQUEST identified some 140,054 protein accessions, or some ~ 26,000 gene symbols, proteins or loci, with at least 5 independent correlations. The X!TANDEM algorithm made at least 5 best fit correlations to more than 14,000 protein gene symbols with p-values and FDR corrected q-values of ~ 0.001 or less. Log10 peptide intensity values showed a Gaussian distribution from E8 to E4 arbitrary counts by quantile plot, and significant variation in average precursor intensity across the disease and controls treatments by ANOVA with means compared by the Tukey–Kramer test. STRING analysis of the top 2000 gene symbols showed a tight association of cellular proteins that were apparently present in the plasma as protein complexes with related cellular components, molecular functions and biological processes. Conclusions The random and independent sampling of pre-fractionated blood peptides by LC-ESI-MS/MS with SQL Server-R analysis revealed the largest plasma proteome to date and was a practical method to quantify and compare the frequency or log10 intensity of individual proteins cleaved ex vivo across populations of plasma samples from multiple clinical locations to discover treatment-specific variation using classical statistics suitable for clinical science. It was possible to identify and quantify nearly all human proteins from EDTA plasma and compare the results of thousands of LC–ESI–MS/MS experiments from multiple clinical populations using standard database methods in SQL Server and classical statistical strategies in the R data analysis system.

scholarly journals Plasma Peptidome

2021 ◽  
Author(s):  
Jaimie Dufresne ◽  
Pete Bowden ◽  
Thanusi Thavarajah ◽  
Angelique Florentinus-Mefailoski ◽  
Zhuo Zhen Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ex Vivo ◽  
Sql Server ◽  
Plasma Samples ◽  
Tryptic Peptides ◽  
Esi Ms ◽  
Human Proteins ◽  
Edta Plasma ◽  
Best Fit ◽  
Gene Symbols

Background It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC–ESI–MS/MS to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations. Methods A systematic method for the organic fractionation of plasma peptides was applied to identify and quantify the endogenous tryptic peptides from human plasma from multiple institutions by C18 HPLC followed nano electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a linear quadrupole ion trap. The endogenous tryptic peptides, or tryptic phospho peptides (i.e. without exogenous digestion), were extracted in a mixture of organic solvent and water, dried and collected by preparative C18. The tryptic peptides from 6 institutions with 12 different disease and normal EDTA plasma populations, alongside ice cold controls for pre-analytical variation, were characterized by mass spectrometry. Each patient plasma was precipitated in 90% acetonitrile and the endogenous tryptic peptides extracted by a stepwise gradient of increasing water and then formic acid resulting in 10 sub-fractions. The fractionated peptides were manually collected over preparative C18 and injected for 1508 LC–ESI–MS/MS experiments analyzed in SQL Server R. Results Peptides that were cleaved in human plasma by a tryptic activity ex vivo provided convenient and sensitive access to most human proteins in plasma that show differences in the frequency or intensity of proteins observed across populations that may have clinical significance. Combination of step wise organic extraction of 200 μL of plasma with nano electrospray resulted in the confident identification and quantification ~ 14,000 gene symbols by X!TANDEM that is the largest number of blood proteins identified to date and shows that you can monitor the ex vivo proteolysis of most human proteins, including interleukins, from blood. A total of 15,968,550 MS/MS spectra ≥ E4 intensity counts were correlated by the SEQUEST and X!TANDEM algorithms to a federated library of 157,478 protein sequences that were filtered for best charge state (2+ or 3+) and peptide sequence in SQL Server resulting in 1,916,672 distinct best-fit peptide correlations for analysis with the R statistical system. SEQUEST identified some 140,054 protein accessions, or some ~ 26,000 gene symbols, proteins or loci, with at least 5 independent correlations. The X!TANDEM algorithm made at least 5 best fit correlations to more than 14,000 protein gene symbols with p-values and FDR corrected q-values of ~ 0.001 or less. Log10 peptide intensity values showed a Gaussian distribution from E8 to E4 arbitrary counts by quantile plot, and significant variation in average precursor intensity across the disease and controls treatments by ANOVA with means compared by the Tukey–Kramer test. STRING analysis of the top 2000 gene symbols showed a tight association of cellular proteins that were apparently present in the plasma as protein complexes with related cellular components, molecular functions and biological processes. Conclusions The random and independent sampling of pre-fractionated blood peptides by LC-ESI-MS/MS with SQL Server-R analysis revealed the largest plasma proteome to date and was a practical method to quantify and compare the frequency or log10 intensity of individual proteins cleaved ex vivo across populations of plasma samples from multiple clinical locations to discover treatment-specific variation using classical statistics suitable for clinical science. It was possible to identify and quantify nearly all human proteins from EDTA plasma and compare the results of thousands of LC–ESI–MS/MS experiments from multiple clinical populations using standard database methods in SQL Server and classical statistical strategies in the R data analysis system.

Mg++ - and Ca++-Induced Platelet Aggregation and ADP

1970 ◽  
Vol 24 (03/04) ◽  
pp. 432-437 ◽  
Author(s):  
S Cronberg ◽  
J. P Caen
Keyword(s):  
Platelet Aggregation ◽  
Pyruvate Kinase ◽  
Pyruvic Acid ◽  
Active Principle ◽  
Platelet Suspension ◽  
Edta Plasma ◽  
Phosphoenol Pyruvic Acid

SummaryReports on platelet aggregation after addition of calcium or magnesium to EDTA- PRP or platelet suspensions were confirmed. An aggregating principle was found in the EDTA-plasma and the supernatant of the platelet suspensions. Aggregation by magnesium in a platelet suspension was inhibited by adenosine and phosphoenol- pyruvic acid and pyruvate kinase, which suggested that the active principle was identical with ADP. Degradation of ADP in EDTA plasma was blocked.It thus appears that aggregation induced by calcium or magnesium in EDTA-PRP and platelet suspension was due to accumulation of spontaneously liberated ADP, which was not degraded.

Zum Mechanismus der Thrombozytenaggregation

1967 ◽  
Vol 18 (03/04) ◽  
pp. 674-685 ◽  
Author(s):  
Th Pfleiderer ◽  
R Brossmer
Keyword(s):  
Edta Plasma

ZusammenfassungBestimmungen der Thrombozytenklebrigkeit in vitro zeigen, daß Zusatz von positiv und negativ geladenen Makromolekülen zu einer Verklumpung von menschlichen Blutplättchen führt. Substanzen mit der gleichen Grundstruktur, aber ohne geladene Gruppen besitzen keine derartige Wirkung. Die Ladung ist für die Aggregation also von entscheidender Bedeutung. Die Verklumpung von Thrombozyten durch geladene Makromoleküle ist nicht wesentlich temperaturabhängig, d.h. sie erfolgt bei 37° C und bei 4° C in gleicher Weise. Es scheint fraglich, ob Stoffwechselprozesse oder enzymatische Vorgänge für den Ablauf der Aggregation von Bedeutung sind. Zwischen der Wirkung positiv und negativ geladener Makromoleküle läßt sich ein prinzipieller Unterschied nach weisen : negativ geladene Substanzen sind in kalziumfreiem Milieu (EDTA-Plasma) nicht mehr wirksam; DEAE-Dextran und Polylysin als Vertreter von positiv geladenen Makromolekülen steigern dagegen in EDTA- und Zitratmilieu gleichermaßen die Klebrigkeit. Das bedeutet, daß Kalziumionen und ADP an diesem Effekt nicht beteiligt sind. Anhand der Befunde werden Vorstellungen zum Mechanismus der Aggregation menschlicher Blutplättchen in vitro entwickelt und deren Bedeutung für Verhältnisse in vivo diskutiert.

C-Terminal amino acid sequence of chicken pepsinogen and its homology with sequences of other acid proteases

1980 ◽  
Vol 45 (4) ◽  
pp. 1144-1154 ◽  
Author(s):  
Miroslav Baudyš ◽  
Helena Keilová ◽  
Vladimír Kostka
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
The Other ◽  
Terminal Sequence ◽  
Terminal Part ◽  
Terminal Amino Acid ◽  
Tryptic Peptides ◽  
Terminal Amino ◽  
High Degree ◽  
Terminal Amino Acid Sequence

To determine the primary structure of the C-terminal part of the molecule of chicken pepsinogen the tryptic, chymotryptic and thermolytic digest of the protein were investigated and peptides derived from this region were sought. These peptides permitted the following 21-residue C-terminal sequence to be determined: ...Ile-Arg-Glu-Tyr-Tyr-Val-Ile-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ser-Pro-Leu-Ser.COOH. A comparison of this structure with the C-terminal sequential regions of the other acid proteases shows a high degree of homology between chicken pepsinogen and these proteases (e.g., the degree of homology with respect to hog pepsinogen and calf prochymosin is about 66%). Additional tryptic peptides, derived from the N-terminal part of the zymogen molecule whose amino acid sequence has been reported before, were also obtained in this study. This sequence was extended by two residues using an overlapping peptide. An ancillary result of this study was the isolation of tryptic peptides derived from other regions of the zymogen molecule.

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