Sensitive Top-Down Proteomics Analysis of a Low Number of Mammalian Cells Using a Nanodroplet Sample Processing Platform

Top-down
proteomics is a powerful tool for characterizing genetic
variations and post-translational modifications at intact protein
level. However, one significant technical gap of top-down proteomics
is the inability to analyze a low amount of biological samples, which
limits its access to isolated rare cells, fine needle aspiration biopsies,
and tissue substructures. Herein, we developed an ultrasensitive top-down
platform by incorporating a microfluidic sample preparation system,
termed nanoPOTS (nanodroplet processing in one pot for trace samples),
into a top-down proteomic workflow. A unique combination of a nonionic
detergent dodecyl-β-d-maltopyranoside (DDM) with urea
as protein extraction buffer significantly improved both protein extraction
efficiency and sample recovery. We hypothesize that the DDM detergent
improves protein recovery by efficiently reducing nonspecific adsorption
of intact proteins on container surfaces, while urea serves as a strong
denaturant to disrupt noncovalent complexes and release intact proteins
for downstream analysis. The nanoPOTS-based top-down platform reproducibly
and quantitatively identified ∼170 to ∼620 proteoforms
from ∼70 to ∼770 HeLa cells containing ∼10 to
∼115 ng of total protein. A variety of post-translational modifications
including acetylation, myristoylation, and iron binding were identified
using only less than 800 cells. We anticipate the nanoPOTS top-down
proteomics platform will be broadly applicable in biomedical research,
particularly where clinical specimens are not available in amounts
amenable to standard workflows.