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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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Tight regulation of the single-cell proteome in X. laevis eggs. (A) Overview of the workflow used to detect technical and biological variation in single cell proteomics. (B) Unsupervised correlation-based clustering of global proteomes mixes technical and biological replicates. See also Supplementary Tables S2 and S3.
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Figure 1: Tight regulation of the single-cell proteome in X. laevis eggs. (A) Overview of the workflow used to detect technical and biological variation in single cell proteomics. (B) Unsupervised correlation-based clustering of global proteomes mixes technical and biological replicates. See also Supplementary Tables S2 and S3.

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)

Tight regulation of the single-cell proteome in X. laevis eggs. (A) Overview of the workflow used to detect technical and biological variation in single cell proteomics. (B) Unsupervised correlation-based clustering of global proteomes mixes technical and biological replicates. See also Supplementary Tables S2 and S3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Tight regulation of the single-cell proteome in X. laevis eggs. (A) Overview of the workflow used to detect technical and biological variation in single cell proteomics. (B) Unsupervised correlation-based clustering of global proteomes mixes technical and biological replicates. See also Supplementary Tables S2 and S3.
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.

Show MeSH
Related in: MedlinePlus