TY - JOUR
T1 - Time-of-Flight Fragmentation Spectra Generated by the Proteomic Analysis of Single Human Cells Do Not Exhibit Atypical Fragmentation Patterns
AU - Orsburn, Benjamin C.
N1 - Publisher Copyright:
© 2023 American Chemical Society.
PY - 2023/3/3
Y1 - 2023/3/3
N2 - 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.
AB - 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.
KW - Orbitrap single cell
KW - TIMSTOF single cell
KW - single-cell proteomics
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U2 - 10.1021/acs.jproteome.2c00715
DO - 10.1021/acs.jproteome.2c00715
M3 - Article
C2 - 36700448
AN - SCOPUS:85147210868
SN - 1535-3893
VL - 22
SP - 1003
EP - 1008
JO - Journal of proteome research
JF - Journal of proteome research
IS - 3
ER -