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. 2014 Dec;13(12):3497-506.
doi: 10.1074/mcp.M113.037309. Epub 2014 Sep 15.

A "Proteomic Ruler" for Protein Copy Number and Concentration Estimation Without Spike-In Standards

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Free PMC article

A "Proteomic Ruler" for Protein Copy Number and Concentration Estimation Without Spike-In Standards

Jacek R Wiśniewski et al. Mol Cell Proteomics. .
Free PMC article

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.

Figures

Fig. 1.
Fig. 1.
Analysis of protein abundances in human plasma using the Total Protein Approach. Whole plasma was processed using the multi-enzyme digestion FASP approach with strong anion exchange peptide fractionation before LC-MS/MS analysis as described in “Experimental Procedures.” Quantifications of selected target proteins are indicated as black dots; the reference values (red bars) are from Refs. and . Two isoforms of creatine kinase were identified with one peptide each, for which we provide annotated MS/MS spectra in supplemental Fig. S1.
Fig. 2.
Fig. 2.
A, the proteomic workflow. Cells were counted and lysed in a buffer containing SDS. Protein concentrations in the whole lysates were determined, and 100-μg aliquots of the whole lysates were successively processed in the proteomic reactor (FASP) format. After detergent removal, proteins were consecutively cleaved with endoproteinase LysC and trypsin. The released LysC and tryptic peptides were subjected to proteomic analysis. Next, RNA and DNA were digested, and the released ribo- and deoxyribonucleotides were spectrophotometrically quantified at 260 nm. Protein contents per single cell were calculated from the cell numbers and the protein concentrations. Alternatively, values of protein mass of single cells were obtained from DNA contents and the protein concentrations. B, determination of the efficiency and yield of RNase and DNase cleavages. Aliquots of mouse liver lysates were processed with the FASP method, and the residual high-molecular-weight material was sequentially cleaved with RNase and DNase (labeled “samples digested with DNase and RNase”). The released ribo- and deoxyribonucleotides were quantified spectrophotometrically at 260 nm. To demonstrate the completeness of digestion over the analyzed range, samples were supplemented with constant amounts of 2 μg of purified DNA or RNA prior to sample processing (labeled “samples + 2 μg RNA/DNA digested with DNase/RNase”). To demonstrate the specificity of the initial RNase digestion, samples were supplemented with DNA and digested with RNase (labeled “samples + 2 μg DNA digested with RNase”).
Fig. 3.
Fig. 3.
Estimation of protein mass per cell using two biochemical approaches and the proteomic ruler method. A, the histone proteomic ruler concept. The mass of cellular DNA is approximately equal to the protein mass of histones. Relating the histone MS signal to the total MS signal therefore allows one to estimate the protein mass per cells at a given cell ploidy and genome size. This method requires neither cell counting nor the determination of protein concentration. B, C, comparison of the values of total protein per cell obtained based on cell counting, DNA determination, and the histone proteomic ruler method. D, cell sizes obtained from retrospective analysis of published proteome datasets of CD4 or CD8a positive or double negative (DN) dendritic cell subtypes and plasmacytoid dendritic cells (pDCs) (36). All values represent the mean of two (cell counting) or three replicates (DNA and histone proteomic ruler quantifications) ± S.D.
Fig. 4.
Fig. 4.
The contribution of PTMs to the estimated total protein content of histones. Comparison of the fractions of the MS signals of individual histones, accumulated by histone type, derived by including different combinations of variable modifications in the database search. A, no variable PTMs (except for the default methionine oxidation and N-terminal acetylation). B, lysine acetylation and serine/threonine/tyrosine phosphorylation. C, lysine mono-, di-, and trimethylation in addition to the modifications searched in B. Comparison of the sum of all histone MS signals without PTMs (from A) and with all PTMs (from C). D, histone MS signal fraction as a function of the depth of analysis, simulated by intensity-based ranking of peptides.
Fig. 5.
Fig. 5.
Comparison of absolute protein abundances calculated using the spike-in and proteomic ruler approaches. A, comparison of protein copy numbers of selected proteins in HeLa cells obtained using spiked-in protein fragments (PrESTs) of known quantities and isotopic label quantification (11) to those calculated using the label-free histone proteomic ruler method. Values represent the mean of three replicates ± S.D. B, comparison of the numbers of peptides overlapping with the PrEST standard used for the SILAC quantification and the total number of peptides used for the proteomic ruler quantification. The deviations of the label-free values from the PrEST-SILAC values are represented as the sizes of the points. C, D, label-free protein copy number estimates correlate with the composition of protein complexes. C, pyruvate dehydrogenase complex. D, TRiC chaperonin.
Fig. 6.
Fig. 6.
Application of the histone proteomic ruler to the global characterization of proteomes. A, average total protein mass per cell. B, average molecular masses of proteins. Values represent the mean of three replicates ± S.D. C, D, abundant proteins tended to be smaller than low-abundance proteins. Motorproteins and filaments were notable exceptions in skeletal muscle.

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