Abstract
Recent work detailed the unique characteristics of fragmentation spectra derived from peptides from single human cells. This valuable report utilized an ultrahigh-field Orbitrap and directly compared the spectra obtained from high-concentration bulk cell HeLa lysates to those obtained from nanogram dilutions of the same and from nanowell-processed single HeLa cells. The analysis demonstrated marked differences between the fragmentation spectra generated at high and single-cell loads, most strikingly, the loss of high-mass y-series fragment ions. As significant differences exist in the physics of Orbitrap and time-of-flight mass analyzers, a comparison appeared warranted. A similar analysis was performed using isolated single pancreatic cancer cells compared to pools consisting of 100 cells. While a reanalysis of the prior Orbitrap data supports the author's original findings, the same trends are not observed in time-of-flight mass spectra of peptides from single human cells. The results are particularly striking when directly comparing the matched intensity fragment values between bulk and single-cell data generated on the same mass analyzers. Instrument acquisition files, processed data, and spectrum libraries are publicly available on MASSIVE via accession MSV000090635.