کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5136327 1494007 2017 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
To elute or not to elute in immunocapture bottom-up LC-MS
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
To elute or not to elute in immunocapture bottom-up LC-MS
چکیده انگلیسی


- The protein conformation on beads and in the eluate affects the digestion yield.
- The quantitative yield of signature peptides was reproducible for all conditions.
- On-beads digestion suffers from non-specific binding to antibodies and/or beads.
- Elution may provide better clean-up and improved column lifetime.

Immunocapture-based bottom-up LC-MS is a promising technique for the quantification of low abundant proteins. Magnetic immunocapture beads provide efficient enrichment from complex samples through the highly specific interaction between the target protein and its antibody. In this article, we have performed the first thorough comparison between digestion of proteins while bound to antibody coated beads versus after elution from the beads. Two previously validated immunocapture based MS methods for the quantification of pro-gastrin releasing peptide (ProGRP) and human chorionic gonadotropin (hCG) were used as model systems. The tryptic peptide generation was shown to be protein dependent and influenced by protein folding and accessibility towards trypsin both on-beads and in the eluate. The elution of proteins bound to the beads was also shown to be incomplete. In addition, the on-beads digestion suffered from non-specific binding of the trypsin generated peptides. A combination of on-beads digestion and elution may be applied to improve both the quantitative (peak area of the signature peptides) and qualitative yield (number of missed cleavages, total number of identified peptides, coverage, signal intensity and number of zero missed cleavage peptides) of the target proteins. The quantitative yield of signature peptides was shown to be reproducible in all procedures tested.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography B - Volumes 1055–1056, 15 June 2017, Pages 51-60
نویسندگان
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