Abstract
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 spectrometry (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 avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along with 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 the MS-loading solvent, consuming only 2 μL of sample. We compared the performance of the microfluidic spectrophotometer with a standard device and determined the optimal sample amount for LC-MS/MS analysis on a Q Exactive HF mass spectrometer using a dilution series of a commercial K562 cell digest. A careful evaluation of selected LC and MS parameters allowed us to define 3 μg as an optimal peptide amount to be injected into this particular LC-MS/MS system. Finally, using tryptic digests from human HEK293T cells and showing that injecting equal peptide amounts, rather than approximate ones, result in less variable LC-MS/MS and protein quantification data. The obtained quality improvement together with easy implementation of the approach makes it possible to routinely quantify MS-ready peptides as a next step in daily proteomics quality control.