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Table 4 Advantages and limitations of proteomic technologies in clinical microbiology

From: Proteomics paves the way for Q fever diagnostics

Technical approach Advantages Limitations
MS-based approaches   
Biotyping • MALDI-TOF MS has several applications in diagnosis and clinical microbiology, including the identification of bacteria and characterization of bacterial proteomes • Selection of protein targets for diagnostic, vaccine or therapeutic development • Applicable to a variety of samples: including bacterial colonies, clinical samples such as blood cultures or urine, and environmental samples • Availability of commercial database (includes 1,660 bacteria isolates and 66 cell types) • Low cost and easy handling compared with classical phenotype-based bacterial identification • When several bacterial species are present (such as Streptococcus spp. or anaerobic Gram-negative bacteria in addition to C. burnetii) poor mass-peak signals may result, which may not be distinguished from the signals produced by the culture medium • Limitations in C. burnetii handling (such as level 3 biosafety laboratory needed, difficulties of culturing an intracellular bacteria) • Blood culture is slow (with slow-growing bacteria) or negative (for example in cases of blood-culture-negative endocarditis caused by C. burnetii) and insufficiently sensitive (for example when the patient has previously received antibiotics)
SDS-PAGE coupled to nano-LC proteome identification • In general, better coverage and sensitivity when compared with MALDI-TOF MS • Identification of low molecular weight proteins • Suitable for investigations of PTMs • Requires culture of the pathogen, for C. burnetii this involves labor-intensive purification from eukaryotic cells and processing of samples • Labor-intensive, time-consuming • Requires skilled operators • Costly
Immunoproteomics (2-DE coupled to MALDI-TOF) • Low cost • Resolved proteins contain PTMs that can be visualized on gels (isoforms) • Robust and suitable method for biomarker selection • Can provide an individual profile of reactivity for each patient sample • Can be used with monoclonal antibodies • Requires the culture and purification of C. burnetii in a level 3 biosafety laboratory, and is labor-intensive • Requires large samples • Variable findings can result, depending on culture conditions, strains, technology and operator skills • Differences between 2D stained gels and immunoblots • Limitations with 2-DE in resolving basic membrane proteins, and low and high MW proteins • In general, low genome coverage (5-30% of total predicted ORFs)
Recombinant protein-based approaches  
Protein array • Does not require the culturing or handling of C. burnetii • Miniaturized systems require small amounts of clinical samples (such as 1-2 μl sera) and allows high-throughput screening (>75% of total predicted ORFs) • Low cost, does not require specific operator skills Escherichia coli system produces proteins without their PTMs (phosphorylations, glycolysations), which are known to be antigenic • Misfolded or multimeric proteins may not be recognized • Requires costly laboratory equipment (fluorescent scanner and spot robot)
ELISA • Easy to perform, does not require sophisticated technology • Lower sensitivity than protein array or IPCR
  1. IPCR, immuno-PCR; LC, liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry; ORF, open-reading frame; PTM, post-translational modification.