Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Summary

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

Abstract

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Introduction

Analysis and identification of undigested proteins by mass spectrometry is referred to as the top-down proteomic analysis^1,2,3,4. It is now an established technique that utilizes electrospray ionization (ESI)⁵ and high-resolution mass analyzers⁶, and sophisticated dissociation techniques, e.g., electron transfer dissociation (ETD), electron capture dissociation (ECD)⁷, ultraviolet photo-dissociation (UV-PD)⁸, etc.

The o....

Protocol

1. Microbiological sample preparation

1. Inoculate 25 mL of Luria broth (LB) in a 50 mL conical tube with E. coli O113:H21 strain RM7788 (or another bacterial strain) from a glycerol stock using a sterile 1 µL loop. Cap the tube and pre-culture at 37 °C with shaking (200 rpm) for 4 h.
2. Aliquot 100 µL of pre-cultured broth and spread onto a LB agar plate supplemented with 400 or 800 ng/mL of mitomycin-C. Culture agar plates statically overnight in an incubator at 37 °C.
   CAUTION: STEC strains are pathogenic microorganisms. Perform all microbiological manipulations, beyond culturing, in a BSL-2 biosafety cabinet.
....

Results

Figure 3 (top panel) shows the MS of STEC O113:H21 strain RM7788 cultured overnight on LBA supplemented with 400 ng/mL mitomycin-C. Peaks at m/z 7276, 7337, and 7841 had been identified previously as cold-shock protein C (CspC), cold-shock protein E (CspE), and a plasmid-borne protein of unknown function, respectively³³. The protein ion at m/z 9780 [M+H]⁺ was analyzed by MS/MS-PSD as shown in Figure 3 (bottom panel). The p.......

Discussion

Protocol considerations
The primary strengths of the current protocol are its speed, simplicity of sample preparation, and use of an instrument that is relatively easy to operate, be trained on, and maintain. Although bottom-up and top-down proteomic analysis by liquid chromatography-ESI-HR-MS are ubiquitous and far superior in many respects to top-down by MALDI-TOF-TOF, they require more time, labor, and expertise. Instrument complexity can often affect whether certain instrument platforms are lik.......

Materials

Name	Company	Catalog Number	Comments
4000 Series Explorer software	AB Sciex	Version 3.5.3
4800 Plus MALDI TOF/TOF Analyzer	AB Sciex
Acetonitrile Optima LC/MS grade	Fisher Chemical	A996-1
BSL-2 biohazard cabinet	The Baker Company	SG403A-HE
Cytochrome-C	Sigma	C2867-10MG
Data Explorer software	AB Sciex	Version 4.9
Focus Protein Reduction-Alkylation kit	G-Biosciences	786-231
GPMAW software	Lighthouse Data	Version 10.0
Incubator	VWR	9120973
LB Agar	Invitrogen	22700-025
Luria Broth	Invitrogen	12795-027
Lysozyme	Sigma	L4919-1G
Microcentrifuge Tubes, 2 mL, screw-cap, O-ring	Fisher Scientific	02-681-343
MiniSpin Plus Centrifuge	Eppendorf	22620207
Mitomycin-C (from streptomyces)	Sigma-Aldrich	M0440-5MG
Myoglobin	Sigma	M5696-100MG
Shaker MaxQ 420HP Model 420	Thermo Scientific	Model 420
Sinapinic acid	Thermo Scientific	1861580
Sterile 1 uL loops	Fisher Scientific	22-363-595
Thioredoxin (E. coli, recombinant)	Sigma	T0910-1MG
Trifluoroacetic acid	Sigma-Aldrich	299537-100G
Water Optima LC/MS grade	Fisher Chemical	W6-4