Residual metals cause variability in methionine oxidation measurements in protein pharmaceuticals using LC-UV/MS peptide mapping

Zang, Li; Carlage, Tyler; Murphy, David; Frenkel, Ruth; Bryngelson, Peter; Madsen, Mark T.; Lyubarskaya, Yelena

doi:10.1016/j.jchromb.2012.03.016

Cited by 54 publications

(56 citation statements)

References 21 publications

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“…Under the three PDT conditions, we identified a total of 431 Met-peptides (156, 29 and 322 for conditions I, II, and III, respectively) corresponding to 302 proteins that showed significantly altered Met oxidation (Table S2). Although Met-peptide oxidation in biological samples can be measured directly, some spontaneous oxidation of Met-peptides (~5–10%) can occur during sample preparation 39 . This could lead to underestimation of the change in Met-peptide oxidation (see Table S3 for a detailed illustration).…”

Section: Resultsmentioning

confidence: 99%

Oxidation of protein-bound methionine in Photofrin-photodynamic therapy-treated human tumor cells explored by methionine-containing peptide enrichment and quantitative proteomics approach

Hsieh

Chien

Yang

et al. 2017

Sci Rep

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In Photofrin-mediated photodynamic therapy (PDT), cell fate can be modulated by the subcellular location of Photofrin. PDT triggers oxidative damage to target cells, including the methionine (Met) oxidation of proteins. Here, we developed a new Met-containing peptide enrichment protocol combined with SILAC-based quantitative proteomics, and used this approach to explore the global Met oxidation changes of proteins in PDT-treated epidermoid carcinoma A431 cells preloaded with Photofrin at the plasma membrane, ER/Golgi, or ubiquitously. We identified 431 Met-peptides corresponding to 302 proteins that underwent severe oxidation upon PDT and observed overrepresentation of proteins related to the cell surface, plasma membrane, ER, Golgi, and endosome under all three conditions. The most frequently oxidized Met-peptide sequence was “QAMXXMM-E/G/M-S/G-A/G/F-XG”. We also identified several hundred potential Photofrin-binding proteins using affinity purification coupled with LC-MS/MS, and confirmed the bindings of EGFR and cathepsin D with Photofrin. The enzyme activities of both proteins were significantly reduced by Photofrin-PDT. Our results shed light on the global and site-specific changes in Met-peptide oxidation among cells undergoing Photofrin-PDT-mediated oxidative stress originating from distinct subcellular sites, and suggest numerous potential Photofrin-binding proteins. These findings provide new insight into the molecular targets through which Photofrin-PDT has diverse effects on target cells.

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Section: Resultsmentioning

confidence: 99%

Oxidation of protein-bound methionine in Photofrin-photodynamic therapy-treated human tumor cells explored by methionine-containing peptide enrichment and quantitative proteomics approach

Hsieh

Chien

Yang

et al. 2017

Sci Rep

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“…The sample digestion step for the method is a time consuming process that could take up to 24 hours at 37 C, a step that could potentially induce artifacts 34 such as asparagine deamidation, 8,[35][36][37] N-terminal glutamine cyclization, 38 and methionine oxidation. 39,40 In addition, the long preparation time hampers the high throughput analysis for a large number of samples in forced degradation studies and clone and media selection.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion

Wang

Liu

et al. 2016

mAbs

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Monoclonal antibodies are subjected to a wide variety of post-translational modifications (PTMs) that cause structural heterogeneity. Characterization and control of these modifications or quality attributes are critical to ensure antibody quality and to define any potential effects on the ultimate safety and potency of antibody therapeutics. The biopharmaceutical industry currently uses numerous tools to analyze these quality attributes individually, which requires substantial time and resources. Here, we report a simple and ultrafast bottom-up liquid chromatography-mass spectrometry (uLC-MS) method with 5 min tryptic digestion to simultaneously analyze multiple modifications, including oxidation, deamidation, isomerization, glycation, glycosylation, and N-terminal pyro-glutamate formation, which can occur during antibody production in mammalian cell culture, during purification and/or on storage. Compared to commonly used preparation procedures, this uLC-MS method eliminates assay artifacts of falsely-increased Met oxidation, Asp isomerization, and Asn deamidation, a problem associated with long digestion times in conventional LC-MS methods. This simple, low artifact multi-attribute uLC-MS method can be used to quickly and accurately analyze samples at any stage of antibody drug development, in particular for clone and media selection during cell culture development.

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“…For the purpose of product characterization, results from peptide mapping need to be interpreted with caution as artifacts during lengthy digestion could occur and lead to incorrect conclusions. [7][8][9][10][11] Another important issue to consider is that any correlation between modifications of different sites can be lost after the protein is cleaved into small peptides.…”

Section: Introductionmentioning

confidence: 99%

A new tool for monoclonal antibody analysis

Zhang

Mueller

et al. 2014

mAbs

123

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Residual metals cause variability in methionine oxidation measurements in protein pharmaceuticals using LC-UV/MS peptide mapping

Cited by 54 publications

References 21 publications

Oxidation of protein-bound methionine in Photofrin-photodynamic therapy-treated human tumor cells explored by methionine-containing peptide enrichment and quantitative proteomics approach

Oxidation of protein-bound methionine in Photofrin-photodynamic therapy-treated human tumor cells explored by methionine-containing peptide enrichment and quantitative proteomics approach

Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion

A new tool for monoclonal antibody analysis

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