posted on 2020-03-17, 17:38authored byTeresa
Mendes Maia, An Staes, Kim Plasman, Jarne Pauwels, Katie Boucher, Andrea Argentini, Lennart Martens, Tony Montoye, Kris Gevaert, Francis Impens
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.