Proteomic Applications in Antimicrobial Resistance and Clinical Microbiology Studies

Figures & data

Table 1 Different Techniques in Quantitative Proteomic with the Associated Strengths and Limitations

Techniques	Methods	Strengths	Limitations	Representative References
2DE	Separation on a gel of the protein content of a sample in two dimensions according to mass and charge, gels are stained and spot intensities in samples are compared among different gels	Simple	Involves large amount of sample	[^Citation131]
		Robust	Low throughput
		Suitable for MS analysis	Poor recovery of hydrophobic proteins
			High inter-gel variability
2-DIGE	Measuring three samples per gels, each of them is labelled with a different fluorescent dye, and the intensities of each gel spot for each sample are measured at a wavelength specific for the label	Multiplexing	Expensive Cy dyes	[^Citation28]
		Better quantitation	Poor recovery of hydrophobic proteins
		Minimal gel to gel variation	Difficulty in separating low molecular weight compounds
Gel-free methods
SILAC	Direct isotope labeling of cells	High throughput	Only suitable for tissue culture models	[^Citation38]
	Differential expression pattern	Robust	Costly reagents
	A vital technique for secreted pathways and secreted proteins in cell culture Comprising labeling of the N-terminus and side chain amine groups of proteins	Sensitive and simple	Not applicable to tissue sample
ICAT	Chemical isotope labeling for quantitative proteomics comparing relative protein abundance between two samples.	Selectively isolates peptide	Post-translational modification information is frequently lost; cannot identify proteins with less than eight cysteines	[^Citation39]
		Compatible with any amount of protein
		Complexity of the peptide mixture is reduced
			Large ICAT label (≈500 Da)
Protein microarrays	Binding of a targeted protein in one	Hight throughput	Synthesis of many different probes	[^Citation44]
	Sample to spotted probes on a “forward” microarray; conversely, binding of specific probes to a targeted protein in spotted samples on a “reverse” microarray; detection of bound proteins by direct labelling or by labelled secondary antibodies	Biomarker identity	Necessary; Identity or class of targeted proteins must be known Limited to detection of proteins targeted by the probes
ITRAQ	Isobaric tagging of peptides	Applicable to versatile samples	Expensive reagents	[^Citation42]
	4 or 8 analysis samples can be quantified	Better quantitation	Incomplete labeling
	Simultaneously; The method is based on the covalent labeling of the N-terminus and sidechain amines of peptides from protein digestions with tags of varying mass	Multiplexing	Involves high amount of sample
SELDI-TOF	Selected part of a protein mixture is Bound to a specific chromatographic surface and the rest washed away	High throughput	Unsuitable for high molecular weight	[^Citation51]
MS		Direct application of whole sample	Proteins; Limited to detection of bound proteins; Lower resolution and mass accuracy
		Small amount of starting material
MALDI-TOF	Application of a protein mixture onto	High throughput	Need for sample fractionation of complex	[^Citation50]
MS	A gold plate; desorption of proteins from the plate by laser energy and measurement of the protein masses; comparison of peak intensities between multiple samples		Samples; More starting material needed for sample fractionation; Unsuitable for high molecular weight proteins
LC-MS/MS	Separation of a mixture of peptides (resulting from protein digestion with trypsin) by one-, two- or three- dimensional LC and measurement of peptide masses by MS-MS	Direct identification of several hundred proteins per sample by MS-MS of peptides	Low throughput	[^Citation53]
			Time consuming
			Detection by MS-MS often not comprehensive; Complicating comparison of different samples
ICPL	After labeling of up to four different proteome states the samples can be combined and the complexity reduced by any separation method currently employed in protein chemistry	High-throughput quantitative proteome profiling on a global scale; able to detect to detect post-translational modifications and protein isoforms; applicable to	Isotopic effect of deuterated tags interferes with retention time of the peptides	[^Citation41]
		Protein like tissue extracts or body fluids
SRM	A powerful tandem mass spectrometry method that can be used to monitor target peptides within a complex protein digest with capability to multiplex the measurement of many analytes in parallel	Highly sensitive, quantitatively accurate and highly reproducible	Detection and quantification of non- abundant proteins; Sensitivity is not comparable to immunological assays;	[^Citation45]
		Quantification of post-translational modifications;
			Limited broad scale application because of difficulty in generating high-quality
		Protein detection is relatively rapid
		Enables detection of non-abundant proteins	SRM assay
Label-free	Measuring the relative concentrations of peptide analytes within two or more samples; require the least sample preparation; as a tool to validate preliminary quantitative proteomics experiments	Avoids labeling	Not suitable for low abundant proteins	[^Citation49]
		Involves less amount of sample	Incomplete digestion may introduce error
		Higher proteome coverage	Multiplexed analysis not possible in one experiment;
			High throughput instrumentation

Table 2 Proteomic Studies of Bacterial Antibiotic Resistance Mechanisms

Antibiotic	Pathogens	Physiological effects	Proteome Analysis	Representative References
Cell wall
Vancomycin	Enterococcus faecium	Vancomycin resistance proteins increased; metabolism-related proteins decreased	2-DE and LC-MS/MS	[^Citation156]
Piperacillin/tazobactam	Escherichia coli	Bacterial virulence, antibiotic resistance, DNA protection, and multidrug efflux pump expression associated with resistance	2D-flurorescence difference gel and electrophoresis (2D-DIGE)	[^Citation147]
Carbapenem	Acinetobacter baumannii	Beta-lactamases, energy, and protein production enzymes are upregulated;	2D-DIGE	[^Citation151]
		OmpW and surface antigen downregulated
Penicillin	Streptococcus pyogenes	Growth phase, stress, and fatty acid biosynthesis (FAB) proteins expression altered	Two dimensional gel electrophoresis (2-DE) and tandem mass spectrometry	[^Citation148]
Cell membrane
Colistin	Escherichia coli	Outer membrane proteins, chaperones, protein biosynthesis factors and metabolic enzymes	2-DE and LC-MS/MS	[^Citation167]
Daptomycin	Staphylococcus aureus	Differences in biofilm formation proteins, cell wall-associated targets	iTRAQ and IPG-isoelectric focusing with LC-MS	[^Citation161]
Protein synthesis
Kanamycin	Escherichia coli	Outer membrane protein expression altered. Identification of novel membrane MipA protein involved in antibiotic resistance	2-DE and LC-MS/MS	[^Citation197]
Tetracycline	Acinetobacter baumannii	Outer membrane proteins decreased expression in membrane and increased secretion	2-DE/MS-MS and 1-DE/LC/MS-MS	[^Citation190]
Linezolid	Streptococcus pneumoniae	Metabolism and transport of carbohydrates involved in resistance to linezolid	2-DE and iTRAQ	[^Citation187]
Protein synthesis
Chloramphenicol	Burkholderia thailandensis	Overexpression of efflux pump systems associated with resistance	SDS-PAGE electrophoresis and LC-MS/MS	[^Citation183]
Erythromycin	Streptococcus pneumoniae	Glyceraldehyde-3-phosphate dehydrogenase upregulation in resistant strain	2-DE and LC-MS/MS	[^Citation201]
DNA synthesis
Fluoroquinolones	Pseudomonas aeruginosa	Overexpression of ATP-binding component of ATP binding cassette (ABC)	2-DE and LC-MS/MS	[^Citation206]
Metronidazole	Clostridium difficile	RecA, ferric uptake regulator (Fur), putative nitro reductases and altered expression of stress-related proteins	iTRAQ and 2D-LC-MS/MS	[^Citation212]
RNA synthesis
Rifampicin	Brucella abortus	Alterations in several metabolic processes and secretion mechanisms	2-DE and LC-MS/MS	[^Citation217]

Figure 1 Overview of bacterial antibiotic resistance mechanisms. Antibiotics target essential bacterial processes and structures to inhibit cell growth and/or causing cell death. The major cellular targets for antibiotics include DNA replication (eg, fluoroquinolones), protein synthesis (eg aminoglycosides), cell wall integrity (eg, penicillins) and folic acid metabolism (eg, sulfonamides).

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