Identification of a chloroform-soluble membrane miniprotein in Escherichia coli and its homolog in Salmonella typhimurium

Abstract

Two homologous 29 amino acid-long highly hydrophobic membrane miniproteins were identified in the Bligh-Dyer lipid extracts of Escherichia coli and Salmonella typhimurium using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The amino acid sequences of the proteins were determined by collision-induced dissociation tandem mass spectrometry, in conjunction with a translating BLAST (tBLASTn) search, i.e., comparing the MS/MS-determined protein query sequence against the six-frame translations of the nucleotide sequences of the E. coli and S. typhimurium genomes. Further MS characterization revealed that both proteins retain the N-terminal initiating formyl-methionines. The methodologies described here may be amendable for detecting and characterizing small hydrophobic proteins in other organisms that are difficult to annotate or analyze by conventional methods.

Figure 1

Negative ion ESI mass spectra of the Bligh-Dyer extracts of E. coli (A) and S. typhimurium (B). The insets show the magnified ion peaks for the [M-3H]³⁻ ions of the small 3 kDa proteins.

Figure 2

Positive ion ESI mass spectra showing the [M+3H]³⁺ ions of the small proteins in the Bligh-Dyer extracts of E. coli (A) and S. typhimurium (B). These positive ion mass spectra are averaged from the spectra acquired by LC/MS (during the LC elution times indicated in the figures).

Figure 3

Assignment of partial amino acid sequences based on the collision-induced dissociation MS/MS spectra of the small 3 kDa proteins from E. coli and S. typhimurium. LC/MS/MS spectra of [M+3H]³⁺ ions at m/z 1008.9 (A and C) for E. coli, and m/z 994.9 (B and D) for S. typhimurium. Peaks labeled with i_v, i_L/I and i_H correspond to the immonium ions of valine, leucine/isoleucine and histidine.

Figure 4

Inter-residual cleavage sites observed in the MS/MS spectra of the [M+3H]³⁺ ions at m/z 1008.9 and m/z 994.9 for the small proteins from E. coli and S. typhimurium, respectively.

Figure 5

Prediction of trans-membrane regions in YnhF and YdhP, encoded by adjacent genes. It is possible, given their genomic and membrane locations, that YnhF forms a complex with YdhP, a putative transporter.