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. 2023 Mar 3;22(3):1003-1008.
doi: 10.1021/acs.jproteome.2c00715. Epub 2023 Jan 26.

Time-of-Flight Fragmentation Spectra Generated by the Proteomic Analysis of Single Human Cells Do Not Exhibit Atypical Fragmentation Patterns

Benjamin C Orsburn  1
Affiliations

Time-of-Flight Fragmentation Spectra Generated by the Proteomic Analysis of Single Human Cells Do Not Exhibit Atypical Fragmentation Patterns

Benjamin C Orsburn. J Proteome Res. .

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.

Keywords: Orbitrap single cell; TIMSTOF single cell; single-cell proteomics.

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Conflict of interest statement

The author declares no competing financial interest.

Figures

Figure 1
Figure 1
Histogram of the matched intensity distribution of each analysis to the curated fragmentation spectra in the NIST library. (A) Bulk Orbitrap fragmentation data compared to single-cell fragmentation data. (B) Same for time-of-flight mass spectra.
Figure 2
Figure 2
Histogram demonstrating the ratio of matched fragment intensity values when comparing single-cell fragmentation spectra to bulk data from each instrument. In this metric, 1.0 would represent spectra with a 100% fragment intensity match between bulk and single-cell spectra.
Figure 3
Figure 3
Fragmentation spectra from single cells observed for the peptide SVPTSTVFYPSDGVATEK from transketolase. (A) Orbitrap SCP data (top) compared to the library reference. (B) TIMSTOF SCP data (top) compared to the same reference.
See this image and copyright information in PMC

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