Step-Wise Assessment and Optimization of Sample Handling
Recovery Yield for Nanoproteomic Analysis of 1000 Mammalian Cells
Posted on 2019-07-31 - 21:30
Protein
and peptide adhesion is a major factor contributing to
sample loss during proteomic sample preparation workflows. Sample
loss often has detrimental effects on the quality of proteomic analysis
by compromising protein identification and data reproducibility. When
starting with a low sample amount, only the most abundant proteins
can be identified, which often offers little insights for biological
research. Although the general idea about severe sample loss from
low amount of starting material is widely presumed in the proteomics
field, quantitative assessment on the impact of sample loss has been
poorly investigated. In the present study, we have quantitatively
assessed sample loss during each step of a conventional in-solution
sample preparation workflow using bicinchoninic acid (BCA) and targeted
LC/MS/MS protein and peptide assays. According to our assessment,
for starting materials of ∼1000 mammalian cells, surface adhesion,
along with desalting and speed-vacuum drying steps, all contribute
heavily to sample loss, in particular for low-abundance proteins.
With this knowledge, we have adapted slippery liquid infused porous
surface (SLIPS) treatment, commercial LoBind tubes, and in-line desalting
during sample processing. With these improvements, we were able to
use a conventional in-solution sample handling method to identify
on average 829 proteins with 1000 U2OS osteosarcoma cells (∼100
ng) with 75-min LC/MS/MS runs, an 11-fold increase in protein identification.
Our optimized in-solution workflow is straightforward and also much
less equipment- and technique-demanding than other advanced sample
preparation protocols in the field.
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Wu, Ruilin; Xing, Sansi; Badv, Maryam; Didar, Tohid F.; Lu, Yu (2019). Step-Wise Assessment and Optimization of Sample Handling
Recovery Yield for Nanoproteomic Analysis of 1000 Mammalian Cells. ACS Publications. Collection. https://doi.org/10.1021/acs.analchem.9b02092
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AUTHORS (5)
RW
Ruilin Wu
SX
Sansi Xing
MB
Maryam Badv
TD
Tohid F. Didar
YL
Yu Lu