Cited by 690Advanced Proteomics Techniques and ApplicationsMass Spectrometry Techniques and ApplicationsAdvanced Biosensing Techniques and Applications

The DOI matches its Crossref record; 44 of the 44 references it cites were found in a registry or an index; 2 of the 34 sources its claims cite were checked for support.

A “Proteomic Ruler” for Protein Copy Number and Concentration Estimation without Spike-in Standards

Open access
Wiśniewski, Jacek R., Hein, Marco Y., Cox, Jürgen, Mann, Matthias·2014·Molecular & Cellular Proteomics

Abstract

Absolute protein quantification using mass spectrometry (MS)-based proteomics delivers protein concentrations or copy numbers per cell. Existing methodologies typically require a combination of isotope-labeled spike-in references, cell counting, and protein concentration measurements. Here we present a novel method that delivers similar quantitative results directly from deep eukaryotic proteome datasets without any additional experimental steps. We show that the MS signal of histones can be used as a "proteomic ruler" because it is proportional to the amount of DNA in the sample, which in turn depends on the number of cells. As a result, our proteomic ruler approach adds an absolute scale to the MS readout and allows estimation of the copy numbers of individual proteins per cell. We compare our protein quantifications with values derived via the use of stable isotope labeling by amino acids in cell culture and protein epitope signature tags in a method that combines spike-in protein fragment standards with precise isotope label quantification. The proteomic ruler approach yields quantitative readouts that are in remarkably good agreement with results from the precision method. We attribute this surprising result to the fact that the proteomic ruler approach omits error-prone steps such as cell counting or protein concentration measurements. The proteomic ruler approach is readily applicable to any deep eukaryotic proteome dataset-even in retrospective analysis-and we demonstrate its usefulness with a series of mouse organ proteomes.

Overview

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Written from the paper’s full text.

TL;DR

The paper introduces a label-free “proteomic ruler” that uses histone MS signal as a proxy for DNA amount to estimate total protein per cell and protein copy numbers without spike-in standards or cell counting. It reports agreement with spike-in/SILAC and biochemical estimates across cell lines, plasma, and mouse tissues.

Key findings

  • In four human cell lines, histone peptides accounted for 2.07–4.03% of total MS signal; equating that to 6.5 pg DNA per diploid cell gave protein mass per cell within 1.24 ± 0.29-fold of cell-counting values.
  • Ribosomal protein signal was 3.61–5.27% of total MS signal, and RNA estimates from this signal were within 1.01 ± 0.13-fold of biochemical RNA measurements.
  • Reanalysis of a PrEST-SILAC dataset gave copy numbers deviating on average 1.5-fold from spike-in values; label-free estimates used 19.4 peptides per protein on average versus 4.7 for PrEST-SILAC, and larger deviations occurred when PrEST-SILAC used ≤2 peptides or label-free used ≤11 peptides.
  • In mouse organs, ovary cells had 42 pg protein per cell and leg muscle about 675 pg per nucleus; liver averaged 464 ± 35 pg per cell, thymus 59 ± 31 pg, and dendritic cell subtypes 64 ± 14 to 95 ± 25 pg.

Methods

  • Samples included human plasma, four cultured human cell lines (U87-MG, A549, PC-3, Hep-G2), and mouse tissues; proteins were processed by FASP with LysC/trypsin digestion, and DNA/RNA were released by RNase/DNase and measured by A260.
  • LC-MS/MS used Q Exactive with HCD or LTQ Orbitrap with CID; data analyzed in MaxQuant/Andromeda against human and mouse UniProt proteomes, with 1% FDR and a Perseus plugin for absolute abundance calculations.
  • Absolute scaling used the Total Protein Approach and histone proteomic ruler; comparisons were made with cell counting, DNA-based cell number estimates, biochemical RNA/protein measurements, PrEST-SILAC copy numbers, and retrospective dendritic cell proteome data.

Limitations

  • The authors state the method requires eukaryotic whole-cell proteome data with unbiased chromatin representation and sufficient depth; histone fraction stabilized only from about 12,000 peptides onward. They also note that in syncytial muscle the ruler yields protein per nucleus rather than per cell.
  • The authors acknowledge assumptions that allow omission of spike-ins, and they report typical agreement within about a factor of 2 with precision measurements or literature values.
  • Evident from the text: validation is limited to a small set of cell lines, plasma, and selected mouse tissues; accuracy depends on known genome size/ploidy and on the assumed DNA-to-histone mass relationship; the retrospective dendritic cell dataset came from in-gel digestion, which the paper notes is incompatible with direct DNA quantification.

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Integrity signals

Registry

Crossref · 10.1074/mcp.m113.037309

Cites

44 of 44 references verified0 citation errors · 0 not checkable

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Claims the paper makes

27 Own interpretation · 27 Own result · 26 Not checked yet · 10 No basis given · 1 Supported so far · 1 Own method

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  • It may state two separate claims as one. 20
  • Probably not a substantial claim. 9
27 of 27
  • The paper relies on this without support.

    Protein determination reagents can cross-react with non-protein cell components such as thiols, nucleic acids, and phospholipids.

    “An additional, often ignored source of errors is the cross-reactivity of the reagents with non-proteinaceous cell components such as thiols, nucleic acids, and phospholipids.”
  • The paper relies on this without support.

    The summed MS signal from all histone-derived peptides is proportional to cumulative histone mass.

    “This value reflects the cumulative histone mass.”
  • The paper relies on this without support.

    The analyzed cell lines have diploid genomes, so their DNA mass per cell is 6.5 pg.

    “We used a value of 6.5 pg for a diploid human cell to calculate cell numbers.”
  • The authors state this as an assumption.

    A protein's cumulative mass in the proteomic sample is directly proportional to the MS signals summed over all peptides derived from it.

    “our copy number calculation assumes a direct proportionality between a protein's cumulative mass in the proteomic sample and the MS signals summed over all peptides derived from it (see Eq. 3).”
  • The paper relies on this without support.

    Many obligate protein complexes are well characterized in terms of composition and stoichiometry, with subunits expressed at equimolar levels.

    “Many obligate protein complexes are well characterized in terms of their composition and stoichiometry with subunits expressed at equimolar levels.”
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Metadata

Authors
Wiśniewski, J. R., Hein, M. Y., Cox, J., & Mann, M.
Affiliations
Max Planck Institute of Biochemistry (DE)
Corresponding author
  • Wiśniewski, Jacek R.
  • Mann, Matthias
Venue
Molecular & Cellular Proteomics
Publisher
Elsevier BV
Published
2014-12
Volume
13
Issue
12
Pages
3497-3506
ISSN
1535-9476 (ISSN-L)
OpenAlex
W2152983548
Cited by
690
References
44
Citations by year
2026: 58 · 2025: 77 · 2024: 82 · 2023: 67 · 2022: 69 · 2021: 57 · 2020: 73 · 2019: 40 · 2018: 62 · 2017: 39 · 2016: 35 · 2015: 25 · 2014: 5
FWCI
14.86
Type
journal article
Language
en
Open access
Locations
  • Molecular & Cellular Proteomics (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Max Planck Digital Library (submittedVersion, other-oa)
  • MPG.PuRe (Max Planck Society) (submittedVersion, other-oa)
Funders
  • European Commission (201648)
  • Max-Planck-Gesellschaft
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced Biosensing Techniques and Applications
Keywords
absolute protein quantification, histones, DNA content
MeSH terms
Amino Acids, Animals, Cell Count, DNA, Histones, Humans, Isotope Labeling, Reference Standards, Gene Expression, Proteome, Cell Line, Tumor, Mice

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