Comparative Proteomics Enables Identification of Nonannotated Cold Shock Proteins in E. coli

Abstract

Recent advances in mass spectrometry-based proteomics have revealed translation of previously nonannotated microproteins from thousands of small open reading frames (smORFs) in prokaryotic and eukaryotic genomes. Facile methods to determine cellular functions of these newly discovered microproteins are now needed. Here, we couple semiquantitative comparative proteomics with whole-genome database searching to identify two nonannotated, homologous cold shock-regulated microproteins in Escherichia coli K12 substr. MG1655, as well as two additional constitutively expressed microproteins. We apply molecular genetic approaches to confirm expression of these cold shock proteins (YmcF and YnfQ) at reduced temperatures and identify the noncanonical ATT start codons that initiate their translation. These proteins are conserved in related Gram-negative bacteria and are predicted to be structured, which, in combination with their cold shock upregulation, suggests that they are likely to have biological roles in the cell. These results reveal that previously unknown factors are involved in the response of E. coli to lowered temperatures and suggest that further nonannotated, stress-regulated E. coli microproteins may remain to be found. More broadly, comparative proteomics may enable discovery of regulated, and therefore potentially functional, products of smORF translation across many different organisms and conditions.

Keywords: E. coli; cold shock; genomics; label-free quantitation; microprotein; non-AUG start codon; proteogenomics; proteomics; small open reading frame; stress response.

Figure 1

Quantitative proteomic gene discovery in E. coli. (A) Schematic overview of the quantitative proteomics protocol. (B) Comparative analysis of nonannotated gene expression begins with parallel preparation of size-selected small proteome samples from control and experimental (cold shock) cells. (C) Nonannotated peptides are sequenced by searching their tandem mass spectrometry (MS/MS) spectra against a six-frame translation of the E. coli genome and excluding sequences matching known proteins. (D) Analysis of the peak area for the respective peptides in the extracted ion chromatograms (EICs) from MS₁ spectra is used to quantify the level of upregulation relative to the control.

Figure 2

Detection and semiquantitative analysis of nonannotated E. coli proteins. (A) Extracted ion chromatograms (EICs) from MS₁ spectra corresponding to (B) MS/MS spectra of nonannotated tryptic peptides identified in our shotgun profiling experiments. The EIC intensity at the same retention time for a 1 Da window around the parent ion mass was compared for the control (red) vs cold shock (blue) samples. Each matched EIC pair is presented on the same y-axis scale. Because the analysis is semiquantitative, substantial intensity in both samples was taken to indicate similar expression. MS/MS spectra (right) presented correspond to the experimental EICs shown (left). Y- and b-ions are shown in red and indicated on the matched peptide scores above each spectrum. m/z, mass to charge ratio. Additional peptides corresponding to each protein as well as scores, precursor mass errors, and charge states corresponding to the MS/MS spectra in this figure can be found in Table S1.

Figure 3

Gene locus diagrams for nonannotated E. coli proteins YmcF (A), YnfQ (B), YnaL (C), and YhiY (D). Line represents chromosomal DNA, annotated protein-coding sequences are represented by gray boxes, and newly reported coding sequences are represented by blue boxes. Arrows indicate 5′–3′ directionality of the coding sequence. Sizes are proportional to length, and genomic coordinates of novel protein sequences are provided. Sizes of novel proteins were calculated either from the first in-frame ATG to stop codon (C, D) or from experimentally determined non-ATG start codons (A, B, vide infra).

Figure 4

Confirmation of expression and cold shock upregulation of novel small proteins. (A) E. coli MG1655 strains with SPA epitope tags added to the C-termini of YmcF, YnfQ, and YhiY were generated. Cell lysates of strains expressing genomically tagged YmcF, YnfQ, and YhiY proteins at 37 °C, 10 °C (cold shock), and 42 °C (heat shock) were separated on a 4–20% SDS gel and stained with Coomassie blue (right). The same samples were also subjected to western blotting (left) and probed with an anti-FLAG antibody. The bands indicated by a red asterisk correspond to YmcF, YnfQ, and YhiY. (B) Bands from the blot were quantified by densitometry, and results are plotted to represent the fold change in expression for the three proteins at 10 °C (cold shock) relative to 37 °C. Error bars were calculated from three biological replicates and represent the standard error of the mean.

Figure 5

Translation of YmcF initiates at an ATT start codon. (A) To verify the translation initiation site for ymcF, a cspG(ds) ymcF plasmid was cloned with a His₆ tag in-frame at the C-terminus of the ymcF coding sequence. In this construct, the cspG start codon was mutated (delete start, ds) to abolish initiation of CspG. We then individually mutated candidate near-cognate ymcF start codons to stop codons. As a negative control, a stop codon was inserted before the His₆ tag in the cspG(ds) ymcF plasmid. Nucleotide numbering starts immediately after the stop codon of cspG, and the sequence is provided in Figure S5. To observe expression, these constructs were introduced into BL21 cells, and IPTG induced cell lysates were subjected to SDS-PAGE followed by blotting against an antibody to the His₆ tag. The YmcF protein band (carat) does not appear when the A88TT codon or the next proceeding near-cognate start codon (G156TG) is mutated to a stop codon. Nucleotide sequence and numbering for ymcF are provided in Figure S5, and additional ymcF mutagenesis experiments are presented in Figure S6. (B) Mutating the A88TT codon in ymcF to ATG results in increased expression of the same major protein product (carat).

Figure 6

Homology and conservation of YmcF and YnfQ. (A) Amino acid sequence alignment of YmcF and YnfQ proteins using Clustal Omega. The two proteins exhibit 66% sequence identity. (B) Nucleotide sequence alignments of ymcF (starting from position −3 relative to the first coding nucleotide) to homologous sequences from Shigella sonnei strain FORC_011 and Salmonella enterica subsp. enterica serovar Anatum str. USDA-ARS-USMARC-1677, which were identified using NCBI BLAST. The ATT start codon is underlined in red.