کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5131615 1491321 2017 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Characterization of succinimide stability during trypsin digestion for LC-MS analysis
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Characterization of succinimide stability during trypsin digestion for LC-MS analysis
چکیده انگلیسی

LC-MS peptide mapping is the most commonly used method to analyze protein modifications. The proteins are generally digested using trypsin at a slightly basic pH at 37 °C from several hours to overnight. Assay-induced artifacts can be generated during this procedure, potentially causing false-positive or false-negative results for a given modification. Unfortunately, for the analysis of succinimide, both false-negative and false-positive results can be generated within the same procedure. This study evaluates the stability of succinimide during the peptide mapping procedure and has demonstrated that up to 13% of pre-existing succinimide was lost during a 4 h trypsin digestion at pH 5.0 which was previously determined to be optimal for the detection of succinimide. The same procedure was able to simultaneously generate approximately 3% succinimide. Using the optimized procedure, it was also found that two aspartate residues that are followed by glycine residues in the conserved Fc region of a recombinant monoclonal antibody were not prone to isomerization. On the other hand, an aspartate residue followed by a glycine in the heavy chain variable domain was highly susceptible to isomerization. Interestingly, the antibody containing the succinimide eluted from an SEC column after the monomer peak.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 526, 1 June 2017, Pages 1-8
نویسندگان
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