Mass spectrometry-based procedure for the identification of ovine casein heterogeneity

2001 ◽  
Vol 68 (1) ◽  
pp. 35-51 ◽  
Author(s):  
PASQUALE FERRANTI ◽  
ROSA PIZZANO ◽  
GIUSEPPINA GARRO ◽  
SIMONETTA CAIRA ◽  
LINA CHIANESE ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Analytical Techniques ◽  
Reversed Phase ◽  
Ionization Mass ◽  
Esi Ms ◽  
On Line ◽  
Protein Components ◽  
Rapid Characterization ◽  
A Minor ◽  
Reference Tools

The efficiency of reversed-phase HPLC, capillary electrophoresis (CE), PAGE and isoelectric focusing with immunoblotting in separating ovine caseins has been evaluated. The assessment was carried out by employing electrospray ionization–mass spectrometry (ESI–MS) and matrix-assisted laser desorption ionization–time of flight as reference tools for identifying protein components. Ovine casein was fractionated by HPLC into four major peaks. With ESI–MS, each peak contained components belonging to only one of the four casein families. On-line liquid chromatography–ESI–MS allowed us to determine each fraction's composition by detecting thirteen αs1-, eleven αs2-, seven β-, and three κ-casein (CN) components. The αs1-CN and αs2-CN consisted of eight and two protein chains respectively of lengths differing through the deletion of one or more peptide sequences; they were also discretely phosphorylated as κ-CN and β-CN. By CE at pH 2·5, each casein fraction was as heterogeneous as that resulting from ESI–MS for the single HPLC-derived fractions. The separation of αs1-CN and αs2-CN proved to be excellent, with the exception of a co-migration of κ0-CN with a minor αs1-CN component and of a glycosylated κ-CN form with low-phosphorylated αs1-CN and β-CN components. Dephosphorylation of whole casein was used to reduce the heterogeneity of the native fractions and by applying currently used analytical techniques it was possible to visualize the protein moiety difference along the CE profile. CE, HPLC, and immunoblotting were all equally capable of effecting an accurate separation of the four dephosphorylated casein families. The spectra obtained by ESI–MS directly on dephosphorylated whole ovine casein samples contained the signals of the four casein families and the relative αs1-CN variants, the non-allelic αs1-CN and αs2-CN forms, dimeric κ-CN and other newly formed peptides. We suggest using this procedure for rapid characterization of whole casein.

scholarly journals Photodegradation of Riboflavin under Alkaline Conditions: What can Gas-Phase Photolysis Tell Us About What Happens in Solution?

2021 ◽  
Author(s):  
Natalie G.K. Wong ◽  
Chris Rhodes ◽  
Caroline E.H. Dessent
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Direct Method ◽  
Photochemical Reactions ◽  
Solution Phase ◽  
Ionization Mass ◽  
Phase Reaction ◽  
Esi Ms ◽  
Alkaline Conditions ◽  
On Line

The application of electrospray ionization mass spectrometry (ESI-MS) as a direct method for detecting reactive intermediates is a technique of developing importance in the routine monitoring of solution-phase reaction pathways. Here, we utilize a novel on-line photolysis ESI-MS approach to detect the photoproducts of riboflavin in aqueous solution under mildly alkaline conditions. Riboflavin is a constituent of many food products, so its breakdown processes are of wide interest. Our on-line photolysis setup allows for solution-phase photolysis to occur within a syringe using UVA LEDs, immediately prior to being introduced into the mass spectrometer via ESI. Gas-phase photofragmentation studies via laser-interfaced mass spectrometry of deprotonated riboflavin, [RFH], the dominant solution-phase species under the conditions of our study, are presented alongside the solution-phase photolysis. The results obtained illustrate the extent to which gas-phase photolysis methods can inform our understanding of the corresponding solution-phase photochemistry. We determine that the solution-phase photofragmentation observed for [RFH] closely mirrors the gas-phase photochemistry, with the m/z 241 ion being the only major condensed-phase photoproduct. Further gas-phase photoproducts are observed at m/z 255, 212, and 145. The value of exploring both the gas- and solution-phase photochemistry to characterize photochemical reactions is discussed.

Coupling of capillary-channeled polymer (C-CP) fibers for reversed phase liquid chromatography and ESI-MS for the determination of proteins in a urine matrix

2016 ◽  
Vol 8 (48) ◽  
pp. 8410-8419 ◽  
Author(s):  
Lei Wang ◽  
Marissa A. Pierson ◽  
R. Kenneth Marcus
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Reversed Phase ◽  
Ionization Mass ◽  
Reversed Phase Liquid Chromatography ◽  
Esi Ms ◽  
Detection Mode ◽  
Phase Liquid ◽  
Urine Matrix

Electrospray ionization mass spectrometry (ESI-MS) analysis provides a great deal of analytical information as a detection mode when coupled with liquid chromatography (LC) and capillary electrophoresis (CE) separations of proteins.

Electrospray ionization mass spectrometry and on-line capillary zone electrophoresis - mass spectrometry for the characterization of citrate synthase

1999 ◽  
Vol 77 (11) ◽  
pp. 1752-1760 ◽  
Author(s):  
Mark E McComb ◽  
Lynda J Donald ◽  
Hélène Perreault
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Ionization ◽  
Electrospray Ionization Mass Spectrometry ◽  
Citrate Synthase ◽  
Time Of Flight ◽  
Ionization Mass Spectrometry ◽  
Ionization Mass ◽  
Wild Type ◽  
Esi Ms ◽  
On Line

The enzyme citrate synthase from E. coli is a protein with a molecular weight (Mr) of 47 885 Da (wild type). This enzyme has been studied extensively, and its amino acid sequence has been characterized. This model protein has been used in this work for development and validation of methods involving capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS). The Mr determinations were conducted using sample infusion ESI-MS, and the tryptic digestion products of wild-type citrate synthase were characterized by on-line CE-ESI-MS coupled with a sheathless interface. On-line experiments were conducted on two different mass spectrometers, a Quattro-LC triple quadrupole instrument equipped with a Z-SprayTM source (Micromass), and a reflecting time-of-flight (TOF) mass spectrometer built in-house in the Time-of-Flight Laboratory, Department of Physics, University of Manitoba. This is the first article to be written on the interfacing of a Z-SprayTM source with CE. Unmodified fused silica capillaries gold-coated sheathless interfaces were used. The on-line CE separations yielded theoretical plate numbers greater than 104 on average. Selected ion electrophorograms (SIE) of the tryptic peptides recorded on the Quattro-LC displayed S/N ratios ranging from ca. 14 to 120 on raw data. These SIE enabled identification of each peptide. The use of reflecting time-of-flight mass spectrometry (TOFMS) afforded mass resolution values of ca. 6000 (m/deltamFWHM), which enabled isotopic resolution of the peptide components. CE-ESI-MS and CE-ESI-TOFMS experiments enabled the generation of a complete tryptic map of citrate synthase.Key words: capillary electrophoresis, electrospray ionization, mass spectrometry, citrate synthase, tryptic digestion, triple quadrupole analyzer, time-of-flight analyzer.

Heterogeneity of caprine κ-casein macropeptide

2001 ◽  
Vol 68 (2) ◽  
pp. 197-208 ◽  
Author(s):  
F. JAVIER MORENO ◽  
ISIDRA RECIO ◽  
AGUSTÍN OLANO ◽  
ROSINA LÓPEZ-FANDIÑO
Keyword(s):  
Mass Spectrometry ◽  
Acid Phosphatase ◽  
Genetic Polymorphism ◽  
Genetic Variant ◽  
Reversed Phase ◽  
Ionization Mass ◽  
Silent Substitution ◽  
Rp Hplc ◽  
On Line ◽  
Molecular Masses

The heterogeneity of caprine caseinmacropeptide (CMP) was determined by means of treatments with neuraminidase and acid phosphatase and analyses by anion exchange FPLC and reversed-phase (RP)-HPLC, with on-line and off-line electrospray ionization mass spectrometry. The main CMP components were two non-glycosylated and di-phosphorylated forms, as well as two other mono-phosphorylated species, each corresponding to a genetic variant of caprine κ-casein due to the silent substitution Ile/ Val at position 119. Asialo-aglyco mono- and di-phosphorylated forms were found in the ratios 8–14% and 86–92%, respectively. Approximately 36% of caprine CMP was glycosylated. Based on the obtained molecular masses, the occurrence of tri-, di- and monosaccharide-containing di-phosphorylated CMP are reported, assuming that N-acetylgalactosamine, galactose, N-acetyl and N-glycolylneuraminic acids would constitute the main monosaccharides of caprine CMP. CMP microheterogeneity due to the genetic polymorphism was also observed in the glycosylated forms.

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