Sulfur containing amino acids – challenge of accurate quantification

Rampler, Evelyn; Dalik, Thomas; Stingeder, Gerhard; Koellensperger, Gunda

doi:10.1039/c2ja10377j

Cited by 18 publications

(17 citation statements)

References 30 publications

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“…The 32 S 16 O detection limit was similar to the detection limits obtained by using other hyphenated ICPMS techniques for S detection. 24,25 Also, the detection limit of 32 S under the Xe gas mode was also similar to previously reported values. 12,19 Taking into account the sample volume of 2.0 μL, in ICPMS under O2 gas mode the absolute detection limits for vasopressin, oxytocin, and amyloid β were 65, 77, and 110 fmol, respectively.…”

Section: Hyphenation Of Nano Hplc and Icpmssupporting

confidence: 78%

“…The S detection limit as a concentration in this study was similar to, or slightly higher than, that reported in previous papers, but a lower absolute detection limit of S was achieved by the hyphenation of nano HPLC and ICPMS. [18][19][20]25,26 To quantify peptides by using nano HPLC-ICPMS, standard solutions containing 100, 200, 500, and 1000 μg S L -1 of S were prepared from ammonium sulfate and used in an external standard method to determine the S concentration of the peptide by means of flow-injection analysis with a 2-μL sample loop. The S concentration of each peptide was measured with relatively good accuracy (within 1.0%) and precision (<5.0% RSD) under the O2 gas modes (Table 1).…”

Section: Hyphenation Of Nano Hplc and Icpmsmentioning

confidence: 99%

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Quantification of Proteins by Measuring the Sulfur Content of Their Constituent Peptides by Means of Nano HPLC-ICPMS

2014

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8 ICPL (isotope-coded protein label), 9 and iTRAQ (isobaric tag for relative and absolute quantification). 10 These methods require not only a specific antibody, but also an amino acid-specific labeling reagents. New protein quantification methods that do 2014 © The Japan Society for Analytical Chemistry † To whom correspondence should be addressed. E-mail: nfuruta@chem.chuo-u.ac.jp Y. S. present address: Faculty of Life and Environmental Science, Department of Regional Environmental Science, Shimane University, 1060 Nishikawatsu-cho, Shimane, Japan. Quantification of Proteins by Measuring the Sulfur Content of Their Constituent Peptides by Means of Nano HPLC-ICPMSYoshinari SUZUKI, Ayumi NOBUSAWA, and Naoki FURUTA † Faculty of Science and Engineering, Department of Applied Chemistry, Chuo University, Bunkyo, Japan The sulfur (S) concentrations of three peptides were determined by using nano HPLC-ICPMS under a flow of O2 in an octapole reaction cell, and the determined values showed a good agreement with theoretical values. This method was then applied to trypsin-digested peptides from human albumin for protein quantification. Assigning of the number of S atoms in each peptide/peak and the tryptic digestion efficiency were important for protein quantification. The number of S atoms in each peptide/peak was assigned by using verification scores that gave the lowest standard deviation of the peptide S concentration and the highest S recovery. The peptide concentrations were calculated as the ratio of the S concentration/the number of S atoms in the peptide/peak. The tryptic digestion efficiency was calculated as the sum of the S concentration in the mono-peptides divided by the total S concentration in a native polyacrylamide gel electrophoresis (PAGE) band before tryptic digestion. Our result indicates that a protein can be quantified through peptide quantification, after taking into account the tryptic digestion efficiency, via S quantification using ICPMS.

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Section: Hyphenation Of Nano Hplc and Icpmssupporting

confidence: 78%

Section: Hyphenation Of Nano Hplc and Icpmsmentioning

confidence: 99%

Quantification of Proteins by Measuring the Sulfur Content of Their Constituent Peptides by Means of Nano HPLC-ICPMS

2014

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“…Acidic hydrolysis of the pellet resulting from the boiling ethanol extraction of samples of 1, 5, 10, and 15 spheroids pooled was based on a procedure described in detail elsewhere [53,54]. In short, 1 mL 6 M hydrochloric acid (Fluka, TraceSELECT from ≥37%) was added to the pellets, already in the conical screw cap vials (Bioquote Limited, York, United Kingdom); screw caps were tightly closed and the samples were hydrolyzed at 100 • C for 24 h. The hydrolyzed samples were evaporated to dryness and stored at −80 • C until analysis.…”

Section: Acidic Hydrolysis Of the Pellet And Amino Acid Analysismentioning

confidence: 99%

Single Spheroid Metabolomics: Optimizing Sample Preparation of Three-Dimensional Multicellular Tumor Spheroids

et al. 2019

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Tumor spheroids are important model systems due to the capability of capturing in vivo tumor complexity. In this work, the experimental design of metabolomics workflows using three-dimensional multicellular tumor spheroid (3D MTS) models is addressed. Non-scaffold based cultures of the HCT116 colon carcinoma cell line delivered highly reproducible MTSs with regard to size and other key parameters (such as protein content and fraction of viable cells) as a prerequisite. Carefully optimizing the multiple steps of sample preparation, the developed procedure enabled us to probe the metabolome of single MTSs (diameter range 790 ± 22 µm) in a highly repeatable manner at a considerable throughput. The final protocol consisted of rapid washing of the spheroids on the cultivation plate, followed by cold methanol extraction. 13C enriched internal standards, added upon extraction, were key to obtaining the excellent analytical figures of merit. Targeted metabolomics provided absolute concentrations with average biological repeatabilities of <20% probing MTSs individually. In a proof of principle study, MTSs were exposed to two metal-based anticancer drugs, oxaliplatin and the investigational anticancer drug KP1339 (sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]), which exhibit distinctly different modes of action. This difference could be recapitulated in individual metabolic shifts observed from replicate single MTSs. Therefore, biological variation among single spheroids can be assessed using the presented analytical strategy, applicable for in-depth anticancer drug metabolite profiling.

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“…Oxidative treatment and protein hydrolysis is described elsewhere. 10 In short, protein samples were oxidized by performic acid prior to hydrolysis. The performic acid derivatisation reagent was prepared by mixing 36 mL of formic acid, 4 mL of H 2 O 2 and 200 mL of phenol and incubation for 30 min at room temperature until the typical yellow colour of performic acid was reached.…”

Section: Oxidation Of Amino Acids and Subsequent Hydrolysis Of Proteinsmentioning

confidence: 99%

In vivo synthesized ³⁴S enriched amino acid standards for species specific isotope dilution of proteins

Hermann

Møller

Gammelgaard

et al. 2016

J. Anal. At. Spectrom.

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Sulfur containing amino acids – challenge of accurate quantification

Cited by 18 publications

References 30 publications

Quantification of Proteins by Measuring the Sulfur Content of Their Constituent Peptides by Means of Nano HPLC-ICPMS

Quantification of Proteins by Measuring the Sulfur Content of Their Constituent Peptides by Means of Nano HPLC-ICPMS

Single Spheroid Metabolomics: Optimizing Sample Preparation of Three-Dimensional Multicellular Tumor Spheroids

In vivo synthesized ³⁴S enriched amino acid standards for species specific isotope dilution of proteins

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Sulfur containing amino acids – challenge of accurate quantification

Cited by 18 publications

References 30 publications

Quantification of Proteins by Measuring the Sulfur Content of Their Constituent Peptides by Means of Nano HPLC-ICPMS

Quantification of Proteins by Measuring the Sulfur Content of Their Constituent Peptides by Means of Nano HPLC-ICPMS

Single Spheroid Metabolomics: Optimizing Sample Preparation of Three-Dimensional Multicellular Tumor Spheroids

In vivo synthesized 34S enriched amino acid standards for species specific isotope dilution of proteins

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In vivo synthesized ³⁴S enriched amino acid standards for species specific isotope dilution of proteins