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Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs.

Smits AH, Lindeboom RG, Perino M, van Heeringen SJ, Veenstra GJ, Vermeulen M - Nucleic Acids Res. (2014)

Bottom Line: While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies.Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance.Comparison between the single-cell proteome and transcriptome reveal poor expression correlation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands Cancer Genomics Netherlands, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.

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Absolute, deep proteome of individual X. laevis eggs. (A) Schematic representation of the workflow used to obtain an absolute, deep proteome. Both the number of fractions and their corresponding nanoLC gradients were optimized for the low amount of protein obtained from a single X. leavis egg. (B) The absolute abundance of all proteins in X. leavis eggs spans over seven orders of magnitude. The black line corresponds to the mean abundance and the gray plusses represent the standard deviation (five replicates). The dashed line indicates the lowest detected protein of the UPS2 spike-in (unique peptides >2) above which abundance can be accurately determined. The inset represents a typical linear regression curve of the measured iBAQ intensities and the known amounts of UPS2 standard. (C) Distribution of the protein copy numbers in Xenopus eggs. Arbitrary cut-offs define five abundance regions for which significant enriched GO terms (FDR < 0.0005) are plotted above these regions. Note the shoulder of high abundant proteins on the right side of the distribution. See also Supplementary Tables S2 and S4.
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Figure 2: Absolute, deep proteome of individual X. laevis eggs. (A) Schematic representation of the workflow used to obtain an absolute, deep proteome. Both the number of fractions and their corresponding nanoLC gradients were optimized for the low amount of protein obtained from a single X. leavis egg. (B) The absolute abundance of all proteins in X. leavis eggs spans over seven orders of magnitude. The black line corresponds to the mean abundance and the gray plusses represent the standard deviation (five replicates). The dashed line indicates the lowest detected protein of the UPS2 spike-in (unique peptides >2) above which abundance can be accurately determined. The inset represents a typical linear regression curve of the measured iBAQ intensities and the known amounts of UPS2 standard. (C) Distribution of the protein copy numbers in Xenopus eggs. Arbitrary cut-offs define five abundance regions for which significant enriched GO terms (FDR < 0.0005) are plotted above these regions. Note the shoulder of high abundant proteins on the right side of the distribution. See also Supplementary Tables S2 and S4.

Mentions: Whole cell lysates were digested using filter-aided sample preparation (FASP) (30). For absolute quantification a standard range of proteins (UPS2, Sigma) was spiked into the sample (1:4 UPS2 to sample (μg/μg)) (31). For in-depth proteomics we applied the digested samples to strong anion exchange (SAX) (32), and we collected the flow through (FT) and pH11, pH8 and pH2 elutions. The peptides were subjected to Stage-Tip desalting and concentration (33) before mass spectrometry analysis. Samples were applied to online nanoLC-MS/MS, using 4 h gradients. For FASP samples, a 4–26% acetonitrile gradient followed by a step wise increase to 76% acetonitrile was used. For SAX samples, 4–14%, 6–17%, 7–18% and 9–21% acetonitrile gradients followed by a step wise increase to 76% acetonitrile were used for the FT, pH11, pH8 and pH2, respectively. Mass spectra were recorded on a LTQ-Orbitrap-Velos mass spectrometer (Thermo Scientific) using collision-induced dissociation (CID) fragmentation on the top 15 most intense precursor ions (data of Figure 1 and Supplementary Figure S1) or recorded on a Q Exactive mass spectrometer (Thermo Scientific) using higher-energy collisional dissociation (HCD) fragmentation on the top 10 most intense precursor ions (data of Figures 2–4).


Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs.

Smits AH, Lindeboom RG, Perino M, van Heeringen SJ, Veenstra GJ, Vermeulen M - Nucleic Acids Res. (2014)

Absolute, deep proteome of individual X. laevis eggs. (A) Schematic representation of the workflow used to obtain an absolute, deep proteome. Both the number of fractions and their corresponding nanoLC gradients were optimized for the low amount of protein obtained from a single X. leavis egg. (B) The absolute abundance of all proteins in X. leavis eggs spans over seven orders of magnitude. The black line corresponds to the mean abundance and the gray plusses represent the standard deviation (five replicates). The dashed line indicates the lowest detected protein of the UPS2 spike-in (unique peptides >2) above which abundance can be accurately determined. The inset represents a typical linear regression curve of the measured iBAQ intensities and the known amounts of UPS2 standard. (C) Distribution of the protein copy numbers in Xenopus eggs. Arbitrary cut-offs define five abundance regions for which significant enriched GO terms (FDR < 0.0005) are plotted above these regions. Note the shoulder of high abundant proteins on the right side of the distribution. See also Supplementary Tables S2 and S4.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4150773&req=5

Figure 2: Absolute, deep proteome of individual X. laevis eggs. (A) Schematic representation of the workflow used to obtain an absolute, deep proteome. Both the number of fractions and their corresponding nanoLC gradients were optimized for the low amount of protein obtained from a single X. leavis egg. (B) The absolute abundance of all proteins in X. leavis eggs spans over seven orders of magnitude. The black line corresponds to the mean abundance and the gray plusses represent the standard deviation (five replicates). The dashed line indicates the lowest detected protein of the UPS2 spike-in (unique peptides >2) above which abundance can be accurately determined. The inset represents a typical linear regression curve of the measured iBAQ intensities and the known amounts of UPS2 standard. (C) Distribution of the protein copy numbers in Xenopus eggs. Arbitrary cut-offs define five abundance regions for which significant enriched GO terms (FDR < 0.0005) are plotted above these regions. Note the shoulder of high abundant proteins on the right side of the distribution. See also Supplementary Tables S2 and S4.
Mentions: Whole cell lysates were digested using filter-aided sample preparation (FASP) (30). For absolute quantification a standard range of proteins (UPS2, Sigma) was spiked into the sample (1:4 UPS2 to sample (μg/μg)) (31). For in-depth proteomics we applied the digested samples to strong anion exchange (SAX) (32), and we collected the flow through (FT) and pH11, pH8 and pH2 elutions. The peptides were subjected to Stage-Tip desalting and concentration (33) before mass spectrometry analysis. Samples were applied to online nanoLC-MS/MS, using 4 h gradients. For FASP samples, a 4–26% acetonitrile gradient followed by a step wise increase to 76% acetonitrile was used. For SAX samples, 4–14%, 6–17%, 7–18% and 9–21% acetonitrile gradients followed by a step wise increase to 76% acetonitrile were used for the FT, pH11, pH8 and pH2, respectively. Mass spectra were recorded on a LTQ-Orbitrap-Velos mass spectrometer (Thermo Scientific) using collision-induced dissociation (CID) fragmentation on the top 15 most intense precursor ions (data of Figure 1 and Supplementary Figure S1) or recorded on a Q Exactive mass spectrometer (Thermo Scientific) using higher-energy collisional dissociation (HCD) fragmentation on the top 10 most intense precursor ions (data of Figures 2–4).

Bottom Line: While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies.Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance.Comparison between the single-cell proteome and transcriptome reveal poor expression correlation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands Cancer Genomics Netherlands, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.

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Related in: MedlinePlus