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Shotgun analysis of proteins in HeLa cells following treatment with polyamine affinity-based probes

ABSTRACT: HeLa cells were incubated with affinity-based probes, synthetic analogs of endogenous metabolites: putrescine, spermidine, and spermine. Cells were photoirradiated briefly and proteins were extracted, digested with trypsin, and the resulting peptides were labeled with TMT. Proteins were quantified across conditions to assess the effect of polyamine analogs on total protein levels in cells. This dataset is related to dataset PXD058706 (HeLa cell protein interactome of polyamine affinity-based probes).

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Remigiusz Serwa

LAB HEAD: Remigiusz Serwa

PROVIDER: PXD062452 | Pride | 2025-09-22

REPOSITORIES: Pride

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			Action	DRS
	RS-91-Total-SET-A_fr-1.raw	Raw
	RS-91-Total-SET-A_fr-2.raw	Raw
	RS-91-Total-SET-A_fr-3.raw	Raw
	RS-91-Total-SET-A_fr-4.raw	Raw
	RS-91-Total-SET-A_fr-5.raw	Raw

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Project description:DNA sequence is a major determinant of the binding specificity of transcription factors (TFs) for their genomic targets. However, eukaryotic cells often express, at the same time, TFs with highly similar DNA binding motifs but distinct in vivo targets. Currently, it is not well understood how TFs with seemingly identical DNA motifs achieve unique specificities in vivo. Here, we used custom protein binding microarrays to analyze TF specificity for putative binding sites in their genomic sequence context. Using yeast TFs Cbf1 and Tye7 as our case study, we found that binding sites of these bHLH TFs (i.e., E-boxes) are bound differently in vitro and in vivo, depending on their genomic context. Computational analyses suggest that nucleotides outside E-box binding sites contribute to specificity by influencing the 3D structure of DNA binding sites. Thus, local shape of target sites might play a widespread role in achieving regulatory specificity within TF families. Three protein binding microarray (PBM) experiments of Saccharomyces cerevisiae transcription factors were performed. Briefly, the PBMs involved binding GST-tagged yeast transcription factors Cbf1 and Tye7 to double-stranded 44K Agilent microarrays in order to determine their binding specificity for putative DNA binding sites in native genomic context. Briefly, we represent three categories of 30-bp genomic sequences: 1) ChIP-chip bound probes, 2) ChIP-chip unbound probes, and 3) negative control probes. ChIP-chip bound probes corresponded to genomic regions bound in vivo by Cbf1 or Tye7 (ChIP-chip P < 0.005 in rich medium (YPD) (Harbison et al., Nature 2004, PMID 15343339)) contained at least two consecutive 8-mers with universal PBM E-score > 0.35 (Zhu et al., Genome Research 2009, PMID 19158363). All putative binding sites occurred at the same position within the probes on the array. M-bM-^@M-^\ChIP-chip unboundM-bM-^@M-^] probes corresponded to genomic regions with ChIP-chip P > 0.5 and at least two consecutive 8-mers at a more stringent universal PBM E-score threshold of 0.4. Negative control probes corresponded to S. cerevisiae intergenic regions with a maximum 8-mer E-score < 0.3. We also designed probes that contain, within constant flanking regions, all 10-bp sequences that occur within the M-bM-^@M-^\ChIP-chip boundM-bM-^@M-^] probes and contain the E-box CACGTG, but are flanked by synthetic rather than native genomic sequence. Each DNA sequence represented on the array is present in 4 replicate spots. We report the PBM signal intensity for each spot. The PBM protocol is described in Berger et al., Nature Biotechnology 2006 (PMID 16998473).

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Shotgun analysis of proteins in HeLa cells following treatment with polyamine affinity-based probes

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