HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics

Recent developments in mass spectrometry-based
single-cell
proteomics
(SCP) have resulted in dramatically improved sensitivity, yet the
relatively low measurement throughput remains a limitation. Isobaric
and isotopic labeling methods have been separately applied to SCP
to increase throughput through multiplexing. Here we combined both
forms of labeling to achieve multiplicative scaling for higher throughput.
Two-plex stable isotope labeling of amino acids in cell culture (SILAC)
and isobaric tandem mass tag (TMT) labeling enabled up to 28 single
cells to be analyzed in a single liquid chromatography–mass
spectrometry (LC–MS) analysis, in addition to carrier, reference,
and negative control channels. A custom nested nanowell chip was used
for nanoliter sample processing to minimize sample losses. Using a
145-min total LC–MS cycle time, ∼280 single cells were
analyzed per day. This measurement throughput could be increased to
∼700 samples per day with a high-duty-cycle multicolumn LC
system producing the same active gradient. The labeling efficiency
and achievable proteome coverage were characterized for multiple analysis
conditions.