Intact Protein Quantitation Using Pseudoisobaric Dimethyl Labeling

2016 ◽  
Vol 88 (14) ◽  
pp. 7198-7205 ◽  
Author(s):  
Houqin Fang ◽  
Kaijie Xiao ◽  
Yunhui Li ◽  
Fan Yu ◽  
Yan Liu ◽  
...  
Keyword(s):  
Intact Protein ◽  
Protein Quantitation ◽  
Dimethyl Labeling

scholarly journals Intact protein LC–MS for pharmacokinetics

2019 ◽  
Vol 4 (4) ◽  
pp. IPK05
Author(s):  
John F Kellie
Keyword(s):  
Large Mass ◽  
Intact Protein ◽  
Clinical Samples ◽  
Quality Attribute ◽  
Human Brain Tissue ◽  
Indiana University ◽  
Disease Biomarker ◽  
Protein Quantitation ◽  
Northwestern University ◽  
Product Quality Attribute

Biography: John Kellie is currently a GlaxoSmithKline (GSK) fellow in the Bioanalysis, Immunogenicity, and Biomarkers group at GSK. John received his B.Sc. in Biochemistry from Indiana University (USA) and his PhD in Chemistry from Northwestern University (USA) studying under Dr Neil Kelleher. He was a post-doctoral scientist at Eli Lilly and Company, where he developed methods for intact protein quantitation of a Parkinson’s Disease biomarker from human brain tissue. At GSK, John utilizes mass spectrometry for development of novel bioanalytical methods for biotherapeutic and protein quantitation from pre-clinical and clinical samples, with a focus on intact protein and large mass quantitation for pharmacokinetics, catabolism, biotransformation and product quality attribute support. John Kellie speaks to the International Journal of Pharmacokinetics about intact protein LC–MS for pharmacokinetic application.

A Strategy of Plasma Protein Quantitation by Selective Reaction Monitoring of an Intact Protein

2003 ◽  
Vol 75 (24) ◽  
pp. 7008-7014 ◽  
Author(s):  
Qin C. Ji ◽  
Ramona Rodila ◽  
Eric M. Gage ◽  
Tawakol A. El-Shourbagy
Keyword(s):  
Plasma Protein ◽  
Intact Protein ◽  
Reaction Monitoring ◽  
Selective Reaction Monitoring ◽  
Selective Reaction ◽  
Protein Quantitation

Control of Thrombin Mediated Cleavage of Protein S

1986 ◽  
Vol 56 (02) ◽  
pp. 151-154 ◽  
Author(s):  
Christina A Mitchell ◽  
Lena Hau ◽  
Hatem H Salem
Keyword(s):  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Surface Protein ◽  
Protein S ◽  
Intact Protein ◽  
Control Mechanisms ◽  
Physiological Control ◽  
Endothelial Cell Surface ◽  
Thrombin Cleavage ◽  
Cofactor Activity

SummaryThrombin has been shown to cleave the vitamin K dependent cofactor protein S with subsequent loss of its cofactor activity. This study examines the control mechanisms for thrombin cleavage of protein S.The anticoagulant activity of activated protein C (APC) is enhanced fourteen fold by the addition of protein S. Thrombin cleaved protein S is seven fold less efficient than the native protein, and this loss of activity is due to reduced affinity of cleaved protein S for APC or the lipid surface compared to the intact protein.In the absence of Ca++, protein S is very sensitive to minimal concentrations of thrombin. As little as 1.5 nM thrombin results in complete cleavage of 20 nM protein S in 10 min and loss of cofactor activity. Ca++, in concentrations greater than 0.5 mM, will inhibit this cleavage and in the presence of physiological Ca++ concentrations, no cleavage of protein S could be demonstrated in spite of high concentrations of thrombin (up to 1 μM) and prolonged incubations (up to two hours). The endothelial surface protein thrombomodulin is very efficient in inhibiting the cleavage of protein S by thrombin suggesting that any thrombin formed on the endothelial cell surface is unlikely to cleave protein S, thus allowing the intact protein to act as a cofactor to APC.We conclude that the inhibitory effects of Ca++ and thrombomodulin on thrombin mediated cleavage of protein S imply that this event, by itself, is unlikely to represent a physiological control of the activity of protein S.

scholarly journals Synthesis and Matrix Properties of α-Cyano-5-phenyl-2,4-pentadienic Acid (CPPA) for Intact Proteins Analysis by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 6054
Author(s):  
Antonio Monopoli ◽  
Angelo Nacci ◽  
Tommaso R. I. Cataldi ◽  
Cosima D. Calvano
Keyword(s):  
Mass Spectrometry ◽  
Laser Desorption ◽  
Protein Analysis ◽  
Ionization Mass Spectrometry ◽  
Intact Protein ◽  
Ionization Mass ◽  
Laser Desorption Ionization ◽  
Desorption Ionization ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption

The effectiveness of a synthesized matrix, α-cyano-5-phenyl-2,4-pentadienic acid (CPPA), for protein analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in complex samples such as foodstuff and bacterial extracts, is demonstrated. Ultraviolet (UV) absorption along with laser desorption/ionization mass spectrometry (LDI-MS) experiments were systematically conducted in positive ion mode under standard Nd:YLF laser excitation with the aim of characterizing the matrix in terms of wavelength absorption and proton affinity. Besides, the results for standard proteins revealed that CPPA significantly enhanced the protein signals, reduced the spot-to-spot variability and increased the spot homogeneity. The CPPA matrix was successful employed to investigate intact microorganisms, milk and seed extracts for protein profiling. Compared to conventional matrices such as sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA) and 4-chloro-α-cyanocinnamic acid (CClCA), CPPA exhibited better signal-to-noise (S/N) ratios and a uniform response for most examined proteins occurring in milk, hazelnut and in intact bacterial cells of E. coli. These findings not only provide a reactive proton transfer MALDI matrix with excellent reproducibility and sensitivity, but also contribute to extending the battery of useful matrices for intact protein analysis.

scholarly journals Culture Dependent and Independent Analysis of Potential Probiotic Bacterial Genera and Species Present in the Phyllosphere of Raw Eaten Produce

2019 ◽  
Vol 20 (15) ◽  
pp. 3661 ◽  
Author(s):  
Sascha Patz ◽  
Katja Witzel ◽  
Ann-Christin Scherwinski ◽  
Silke Ruppel
Keyword(s):  
Protein Pattern ◽  
Probiotic Bacteria ◽  
Lepidium Sativum ◽  
Intact Protein ◽  
Bacillus Species ◽  
Thymus Vulgaris ◽  
Petroselinum Crispum ◽  
Bacterial Strains ◽  
Fresh Weight ◽  
Trigonella Foenum Graecum L

The plant phyllosphere is colonized by a complex ecosystem of microorganisms. Leaves of raw eaten vegetables and herbs are habitats for bacteria important not only to the host plant, but also to human health when ingested via meals. The aim of the current study was to determine the presence of putative probiotic bacteria in the phyllosphere of raw eaten produce. Quantification of bifidobacteria showed that leaves of Lepidium sativum L., Cichorium endivia L., and Thymus vulgaris L. harbor between 103 and 106 DNA copies per gram fresh weight. Total cultivable bacteria in the phyllosphere of those three plant species ranged from 105 to 108 CFU per gram fresh weight. Specific enrichment of probiotic lactic acid bacteria from C. endivia, T. vulgaris, Trigonella foenum-graecum L., Coriandrum sativum L., and Petroselinum crispum L. led to the isolation of 155 bacterial strains, which were identified as Pediococcus pentosaceus, Enterococcus faecium, and Bacillus species, based on their intact protein pattern. A comprehensive community analysis of the L. sativum leaves by PhyloChip hybridization revealed the presence of genera Bifidobacterium, Lactobacillus, and Streptococcus. Our results demonstrate that the phyllosphere of raw eaten produce has to be considered as a substantial source of probiotic bacteria and point to the development of vegetables and herbs with added probiotic value.

Sign in / Sign up

Export Citation Format

Share Document