Measurements of drug–protein binding by using immobilized human serum albumin liquid chromatography–mass spectrometry

Cheng, Ying; Ho, Elena; Subramanyam, Babu; Tseng, Jih-Lie

doi:10.1016/j.jchromb.2004.06.006

Cited by 54 publications

(132 citation statements)

References 26 publications

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“…When using this approach in qualitative studies, the sequence coverage of HSA has been reported to be 97.4%. 29 However, not all of the peptides detected in this technique have sufficient intensities to allow their use in quantitative proteomics and measurements of 18 O/ 16 O ratios. This additional requirement was found in this current study to lower the effective sequence coverage of HSA to 76.9%; however, this level of coverage was still sufficient for examining most of the possible immobilization sites on HSA.…”

Section: General Considerations In Methods Developmentmentioning

confidence: 99%

“…The reproducibility of the MALDI-TOF MS analysis was examined early in this work by using a mixture of 18 O and 16 O-labeled peptides from tryptic digests of soluble HSA. In one such study, a peptide MH + peak with a mass of 927.4939 had a relative standard deviation of !…”

Section: General Considerations In Methods Developmentmentioning

confidence: 99%

“…Finally, the 18 O/ 16 O ratio for the desired peptide in a mixture of soluble and immobilized protein preparations that have been separately digested in 16 O-or 18 O-enriched water can be calculated by using these results in eq 8. (8) In this report this corrected ratio was then used to determine the relative extent to which a given region on the protein was involved in its immobilization to a solid support.…”

Section: Theorymentioning

confidence: 99%

“…The dried samples were reconstituted in 120 μL of pH 7.8, 50 mM ammonium bicarbonate buffer prepared in standard deionized water (99.8% H 2 16 O) for the immobilized HSA or in 18 O-enriched water (97% H 2 18 O) for the soluble HSA. Both aliquots were combined with 2.0 μg trypsin (giving a substrate-to-enzyme ratio of 30:1), with these mixtures then being incubated at 37 °C for 18 h.…”

Section: Hsa Immobilization and Pretreatmentmentioning

confidence: 99%

“…The sequence coverage for HSA was calculated by using the fraction of all amino acids in HSA that occurred in the detected peptides and that could be identified as arising from a specific portion of this protein's primary sequence. 29 For the quantitative analysis of peptides, the ratio of each 18 O and 16 O-labeled peptide pair in the digest mixtures was determined as described in the THEORY. The pK a values for the lysine residues in HSA were calculated using PROPKA.…”

Section: Mass Spectrometric Analysismentioning

confidence: 99%

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Identification and Quantitative Studies of Protein Immobilization Sites by Stable Isotope Labeling and Mass Spectrometry

Cerny²,

Hage

2006

Anal. Chem.

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A method was developed for characterizing immobilization sites on a protein based on stable isotope labeling and MALDI-TOF mass spectrometry. The model for this work was human serum albumin (HSA) immobilized onto silica by the Schiff base method. The immobilized HSA was digested by various proteolytic enzymes in the presence of normal water, while soluble HSA was digested in 18 O-enriched water for use as an internal standard. These two digests were mixed and analyzed, with the 18 O/ 16 O ratio for each detected peptide then being measured. Several peptides in the tryptic, Lys-C, and Glu-C digests gave significantly higher 18 O/ 16 O ratios than other peptides in the same digests, implying their involvement in immobilization. Analysis of these results led to identification of the N-terminus and several lysines as likely immobilization sites for HSA (e.g., K4, K41, K190, K225, K313 and K317). It was also possible from these results to quantitatively rank these sites in terms of the relative degree to which each might take part in immobilization. This method is not limited to HSA and silica but can be used with other proteins and supports.

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Section: General Considerations In Methods Developmentmentioning

confidence: 99%

Section: General Considerations In Methods Developmentmentioning

confidence: 99%

Section: Theorymentioning

confidence: 99%

Section: Hsa Immobilization and Pretreatmentmentioning

confidence: 99%

Section: Mass Spectrometric Analysismentioning

confidence: 99%

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Identification and Quantitative Studies of Protein Immobilization Sites by Stable Isotope Labeling and Mass Spectrometry

Cerny²,

Hage

2006

Anal. Chem.

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Analysis of Individual Signaling Complexes by mMAPS, a Flow‐Proteometric System

Chou

Tsou

Hsu

et al. 2016

CP Molecular Biology

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Signal transduction is essential for maintaining cells’ normal physiological functions, and deregulation of signaling can lead to diseases such as diabetes and cancers. Some of the major players in signal delivery are molecular complexes composed of proteins and nucleic acids. This unit describes a technique called microchannel for Multi-parameter Analysis of Proteins in a Single-complex or mMAPS for analyzing and quantifying target signaling complexes individually. mMAPS is a flow-proteometric-based system that allows detection of individual proteins or complexes flowing through a microfluidic channel. Specific target proteins and nucleic acids labeled by fluorescent tags are harvested from tissue samples or cultured cells for analysis by the mMAPS system. Overall, mMAPS enables both detection of multiple components within a single complex and direct quantification of different populations of molecular complexes in one setting in a short timeframe, requiring very low sample input.

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Toxicokinetics: An Integral Component of Preclinical Toxicity Studies

Singh¹

2010

Pharmaceutical Sciences Encyclopedia

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Measurements of drug–protein binding by using immobilized human serum albumin liquid chromatography–mass spectrometry

Cited by 54 publications

References 26 publications

Identification and Quantitative Studies of Protein Immobilization Sites by Stable Isotope Labeling and Mass Spectrometry

Identification and Quantitative Studies of Protein Immobilization Sites by Stable Isotope Labeling and Mass Spectrometry

Analysis of Individual Signaling Complexes by mMAPS, a Flow‐Proteometric System

Toxicokinetics: An Integral Component of Preclinical Toxicity Studies

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