A simple approach for accurate peptide quantification in MS-based proteomics (original) (raw)
Despite its growing popularity and use, bottom-up proteomics remains a complex analytical methodology. Its general workflow consists of three main steps: sample preparation, liquid chromatography coupled to tandem mass spec-trometry (LC-MS/MS) and computational data analysis. Quality assessment of the different steps and components of this workflow is instrumental to identify technical flaws and to avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along the sample preparation protocol, in particular after proteolytic digestion, is not yet routinely implemented because of the lack of an accurate and straightforward method to quantify peptides. Here, we report on the use of a microfluidic UV/visible spectrophotometer to quantify MS-ready peptides directly in MS loading solvent, consuming only 2 microliter of sample. We determined the optimal peptide amount for LC-MS/MS analysis on a Q Exactive HF mass spectrometer using a d...
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