scholarly journals Influence of NanoLC Column and Gradient Length as well as MS/MS Frequency and Sample Complexity on Shotgun Protein Identification of Marine Bacteria

2017 ◽  
Vol 27 (3) ◽  
pp. 199-212 ◽  
Author(s):  
Lars Wöhlbrand ◽  
Ralf Rabus ◽  
Bernd Blasius ◽  
Christoph Feenders
Keyword(s):  
Marine Bacteria ◽  
Protein Identification ◽  
Shotgun Proteomics ◽  
Sample Complexity ◽  
Column Length ◽  
Peptide Separation ◽  
Total Analysis Time ◽  
Total Analysis ◽  
Nano Liquid Chromatography ◽  
Esi Mass Spectrometry

Protein identification by shotgun proteomics, i.e., nano-liquid chromatography (nanoLC) peptide separation online coupled to electrospray ionization (ESI) mass spectrometry (MS)/MS, is the most widely used gel-free approach in proteome research. While the mass spectrometer accounts for mass accuracy and MS/MS frequency, the nanoLC setup and gradient time influence the number of peptides available for MS analysis, which ultimately determine the number of proteins identifiable. Here, we report on the influence of (i) analytical column length (15, 25, or 50 cm) coupled to (ii) the applied gradient length (120, 240, 360, 480, or 600 min), as well as (iii) MS/MS frequency on peptide/protein identification by shotgun proteomics of (iv) 2 marine bacteria. Longer gradients increased the number of peptides/proteins identified as well as the reproducibility of identification. Furthermore, longer analytical columns strictly enlarge the covered proteome complement. Notably, the proteome complement identified with a short column and applying a long gradient is also covered when using longer columns with shorter gradients. Coverage of the proteome complement further increases with higher MS/MS frequency. Compilation of peptide lists of replicate analyses (same gradient length) improves protein identification, while compilation of analyses with different gradient lengths yields a similar or even higher number of proteins using comparable or even less total analysis time.

Breaching the 10 Second Barrier of Total Analysis Time for Complex Matrices via Automated Coated Blade Spray

2019 ◽  
Vol 91 (20) ◽  
pp. 13039-13046 ◽  
Author(s):  
Alexander Kasperkiewicz ◽  
Germán Augusto Gómez-Ríos ◽  
Dietmar Hein ◽  
Janusz Pawliszyn
Keyword(s):  
Analysis Time ◽  
Complex Matrices ◽  
Total Analysis Time ◽  
Total Analysis

Determination of Methyl 2-Benzimidazolecarbamate in Wine by Competitive Inhibition Enzyme Immunoassay

1993 ◽  
Vol 76 (4) ◽  
pp. 851-856 ◽  
Author(s):  
Rodney J Bushway ◽  
Lance R Paradis ◽  
Lewis B Perkins ◽  
Titan S Fan ◽  
Barbara E S Young ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Competitive Inhibition ◽  
White Wines ◽  
Average Recovery ◽  
Total Analysis Time ◽  
Coefficients Of Variation ◽  
Limit Of Quantitation ◽  
Total Analysis ◽  
Commercial Kit

Abstract A benomyl polyclonal enzyme immunoassay (EIA) commercial kit was used to quantitate methyl 2- benzimidazolecarbamate (MBC), a degradation product of benomyl in wine. Total analysis time, including sample preparation, was 30 min. As many as 8 samples can be analyzed simultaneously with a limit of quantitation of 5 ppb. The assay logarithmic response was linear from 0.4 to 26 ppb MBC. Intra-assay percent coefficients of variation (%CVs) ranged from 2.4 to 13 for standards and from 7.4 to 21 for actual wine samples. Interassay %CVs varied from 2.6 to 15 for the standards and from 6.9 to 23 for the samples. Average recovery from samples spiked at 10–10 000 ppb was 93% for evaporated red and white wines. MBC was determined in 134 different wines by immunoassay and liquid chromatography (LC). Of these samples, 98 were positive for MBC by both methods with a correlation coefficient (r) of 0.986. The other 36 samples had MBC levels that either were not detectable by either procedure or were below the 10 ppb limit of quantitation for LC. Concentrations of MBC in wine ranged from 5 to 1329 ppb, with the majority ranging from 10 to 300 ppb. Also, a mini-study was conducted using the plate EIA format.

Optimizing Shotgun Proteomics Analysis for a Confident Protein Identification and Quantitation in Orphan Plant Species: The Case of Holm Oak (Quercus ilex)

2020 ◽  
pp. 157-168
Author(s):  
Isabel Gómez-Gálvez ◽  
Rosa Sánchez-Lucas ◽  
Bonoso San-Eufrasio ◽  
Luis Enrique Rodríguez de Francisco ◽  
Ana M. Maldonado-Alconada ◽  
...  
Keyword(s):  
Plant Species ◽  
Protein Identification ◽  
Quercus Ilex ◽  
Shotgun Proteomics ◽  
Holm Oak ◽  
Proteomics Analysis ◽  
Protein Identification And Quantitation

Characteristic Chromatogram: A Method of Discriminate and Quantitative Analysis for Quality Evaluation of Uncaria Stem with Hooks

Planta Medica ◽  
2017 ◽  
Vol 84 (06/07) ◽  
pp. 449-456 ◽  
Author(s):  
Jinjun Hou ◽  
Ruihong Feng ◽  
Yibei Zhang ◽  
Huiqin Pan ◽  
Shuai Yao ◽  
...  
Keyword(s):  
Quality Evaluation ◽  
Indole Alkaloids ◽  
Total Content ◽  
Traditional Chinese Medicines ◽  
Herbal Drugs ◽  
Chinese Medicines ◽  
Total Analysis Time ◽  
Total Analysis ◽  
Single Standard

AbstractIt remains a challenge to establish new monographs for herbal drugs derived from multiple botanical sources. Specifically, the difficulty involves discriminating and quantifying these herbs with components whose levels vary markedly among different samples. Using Uncaria stem with hooks as an example, a characteristic chromatogram was proposed to discriminate its five botanical origins and to quantify its characteristic components in the chromatogram. The characteristic chromatogram with respect to the components of Uncaria stem with hooks with the five botanical origins was established using 0.02% diethylamine and acetonitrile as the mobile phase. The total analysis time was 50 min and the detection wavelength was 245 nm. Using the same chromatogram parameters, the single standard to determine multicomponents method was validated to simultaneously quantify nine indole alkaloids, including vincosamide, 3α-dihydrocadambine, isocorynoxeine, corynoxeine, isorhynchophylline, rhynchophylline, hirsuteine, hirsutine, and geissoschizine methyl ether. The results showed that only the Uncaria stem with hooks from Uncaria rhynchophylla, the most widely used in the herbal market, showed the presence of these nine alkaloids. The conversion factors were 1.27, 2.32, 0.98, 1.04, 1.00, 1.02, 1.26, 1.33, and 1.25, respectively. The limits of quantitation were lower than 700 ng/mL. The total contents of 31 batches of Uncaria stem with hooks were in the range of 0.1 – 0.6%, except for Uncaria hirsuta Havil and Uncaria sinensis (Oliv.) Havil. The results also showed that the total content of indole alkaloids tended to decrease with an increase in the hook diameter. This showed that the characteristic chromatogram is practical for controlling the quality of traditional Chinese medicines with multiple botanical origins.

scholarly journals Putative Antimicrobial Peptides of the Posterior Salivary Glands from the Cephalopod Octopus vulgaris Revealed by Exploring a Composite Protein Database

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 757 ◽  
Author(s):  
Daniela Almeida ◽  
Dany Domínguez-Pérez ◽  
Ana Matos ◽  
Guillermin Agüero-Chapin ◽  
Hugo Osório ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Salivary Glands ◽  
Protein Identification ◽  
Inflammatory Responses ◽  
Shotgun Proteomics ◽  
Octopus Vulgaris ◽  
Protein Database ◽  
Venom Protein ◽  
Protein Toxin ◽  
Proteomics Approach

Cephalopods, successful predators, can use a mixture of substances to subdue their prey, becoming interesting sources of bioactive compounds. In addition to neurotoxins and enzymes, the presence of antimicrobial compounds has been reported. Recently, the transcriptome and the whole proteome of the Octopus vulgaris salivary apparatus were released, but the role of some compounds—e.g., histones, antimicrobial peptides (AMPs), and toxins—remains unclear. Herein, we profiled the proteome of the posterior salivary glands (PSGs) of O. vulgaris using two sample preparation protocols combined with a shotgun-proteomics approach. Protein identification was performed against a composite database comprising data from the UniProtKB, all transcriptomes available from the cephalopods’ PSGs, and a comprehensive non-redundant AMPs database. Out of the 10,075 proteins clustered in 1868 protein groups, 90 clusters corresponded to venom protein toxin families. Additionally, we detected putative AMPs clustered with histones previously found as abundant proteins in the saliva of O. vulgaris. Some of these histones, such as H2A and H2B, are involved in systemic inflammatory responses and their antimicrobial effects have been demonstrated. These results not only confirm the production of enzymes and toxins by the O. vulgaris PSGs but also suggest their involvement in the first line of defense against microbes.

scholarly journals Rapid Gas Chromatographic Method for Simultaneous Determination of Cholesterol and α-Tocopherol in Eggs

1998 ◽  
Vol 81 (6) ◽  
pp. 1177-1184 ◽  
Author(s):  
Nickos Botsoglou ◽  
Dimitrios Fletouris ◽  
Ioannis Psomas ◽  
Antonios Mantis
Keyword(s):  
Simultaneous Determination ◽  
Chromatographic Method ◽  
Labor Cost ◽  
Precision Data ◽  
Total Analysis Time ◽  
Accuracy And Precision ◽  
Relative Standard ◽  
Total Analysis ◽  
Gas Chromatographic Method

Abstract A new method was developed for simultaneous determination of cholesterol and α-tocopherol in eggs. It involves rapid and simple sample preparation accomplished in one tube and chromatographic separation that does not require derivatization of analytes. Total analysis time per sample is 40 min. Labor, cost, and use of hazardous chemicals are minimized. To ensure selectivity, accuracy, and precision, critical analytical parameters were investigated. Overall recoveries were 98.8 and 99.2% for cholesterol and α-tocopherol, respectively. Linearity was acceptable for both analytes (r = 0.9964 for cholesterol and 0.9996 for α-tocopherol) in the fortification range examined. Precision data based on within-day and between-days variation gave overall relative standard deviations of 2.0% for cholesterol and 7.0% for α-tocopherol.The method was applied successfully for quantitation of cholesterol and α-tocopherol in eggs.

Ascorbic and dehydroascorbic acids measured in plasma preserved with dithiothreitol or metaphosphoric acid

1990 ◽  
Vol 36 (10) ◽  
pp. 1750-1755 ◽  
Author(s):  
S A Margolis ◽  
R C Paule ◽  
R G Ziegler
Keyword(s):  
Ascorbic Acid ◽  
Reference Material ◽  
Rapid Method ◽  
Dehydroascorbic Acid ◽  
Analysis Time ◽  
Plasma Samples ◽  
Metaphosphoric Acid ◽  
Total Analysis Time ◽  
Total Analysis ◽  
The Stability

Abstract We describe a rapid method for accurately and precisely measuring ascorbic acid and dehydroascorbic acid in plasma. Total analysis time is less than 10 min, replicate analyses of a single pool provide precision less than or equal to 2%, and values measured in supplemented samples agree with known concentrations of 4.68 and 11.83 mg/L. The stability and homogeneity of lyophilized plasma samples supplemented with ascorbic acid and dithiothreitol are documented. We also describe a procedure in which metaphosphoric acid (50 g/L) is used to prepare a reference material for the measurement of ascorbic acid and dehydroascorbic acid. The procedure for both acids consists of first measuring the native ascorbic acid, then reducing the dehydroascorbic acid, at neutral pH, with dithiothreitol, and finally measuring the total ascorbic acid; dehydroascorbic acid is then determined by difference. The metaphosphoric-acid-treated samples were stable at -70 degrees C, but stability decreased with temperature over the range examined, 4-50 degrees C.

Simple, reliable chromatographic measurement of oxalate in urine.

1982 ◽  
Vol 28 (7) ◽  
pp. 1457-1460 ◽  
Author(s):  
A Di Corcia ◽  
R Samperi ◽  
G Vinci ◽  
G D'Ascenzo
Keyword(s):  
Polyethylene Glycol ◽  
Limit Of Detection ◽  
Graphitized Carbon Black ◽  
Urinary Oxalate ◽  
Total Analysis Time ◽  
Graphitized Carbon ◽  
Analytical Recovery ◽  
Urine Oxalate ◽  
Total Analysis ◽  
Chromatographic Measurement

Abstract In this assay for oxalate in urine, oxalate is adsorbed from the urine onto graphitized carbon black (Carbopack B). After desorption and removal of the solvent, oxalic acid is gas-chromatographically measured after being derivatized with BF3/methanol. Chromatography is on Carbopack B/polyethylene glycol (Mr 20000), 93.7/6.3 by weight. The lower limit of detection of urinary oxalate is about 3 mg/L (CV less than or equal to 3.6%). A series of oxalate determinations in 24-h urine samples of 15 subjects gave a mean of 47.1 (SD 15.4) mg/24 h, with an analytical recovery of 97.4% (SD 3.0%, range 93.6-102.4%). Total analysis time for one sample is about 2 h.

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