Francisella tularensis causes the zoonotic disease tularemia, and is considered a potential bioterrorist agent due to its extremely low infection dose and potential for airborne transmission. Presently, F. tularensis is divided into four subspecies; tularensis, holarctica, mediasiatica and novicida. Phenotypic discrimination of the closely related subspecies with traditional methods is difficult and tedious. Furthermore, the results may be vague and they often need to be complemented with virulence tests in animals. Here, we have used surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to discriminate between the four subspecies of F. tularensis. The method is based on the differential binding of protein subsets to chemically modified surfaces. Bacterial thermolysates were added to anionic, cationic, and copper ion-loaded immobilized metal affinity SELDI chip surfaces. After binding, washing, and SELDI-TOF-MS different protein profiles were obtained. The spectra generated from the different surfaces were then used to characterize each bacterial strain. The results showed that the method was reproducible, with an average intensity variation of 21%, and that the mass precision was good (300-450 ppm). Moreover, in subsequent cluster analysis and principal component analysis (PCA) data for the analyzed Francisella strains grouped according to the recognized subspecies. Partial least squares-discriminant analysis (PLS-DA) of the protein profiles also identified proteins that differed between the strains. Thus, the protein profiling approach based on SELDI-TOF-MS holds great promise for rapid high-resolution phenotypic identification of bacteria.

We demonstrate a novel method to analyse ex situ prepared protein chips by attenuated total reflection Fourier IR spectroscopy (ATR‐FTIR), which circumvents tedious functionalisation steps of internal reflection elements (IREs), and simultaneously allows for complementary measurements by other analytical techniques. This concept is proven by utilising immobilised metal affinity capture (IMAC™) chips containing about 10 μm thick films of copolymers coated with nitrilotriacetic acid (NTA) groups, which originally was manufactured for surface enhanced laser desorption ionisation (SELDI) spectrometry. Three immobilisation steps were analysed by ATR‐FTIR spectroscopy: 1) NTA complexation with nickel(II) ions 2) binding of two histidine (His)‐tagged synthetic peptides of 25 (25‐His6) and 48 (48‐His6) amino acids to the NTA‐groups and 3) attachment of a ligand, mesyl amide, to the surface‐bound 48‐His6. Despite interference from H2O, both amide I and II were well resolved. Utilising peptide adsorption in the thick copolymer matrix yields a high saturation peptide concentration of ≈100 mg mL−1 and a dissociation constant of 116±11 μM, as determined by a detailed analysis of the Langmuir adsorption isotherm. The mesyl amide ligand was directly seen in the raw ATR‐FTIR spectrum with specific peaks in the fingerprint region at 1172 and 1350 cm−1. Several aspects of the fine structure of the amide I band of the peptide were analysed: influences from secondary structure, amino side chains and competing contamination product. We believe that this approach has great potential as a stand‐alone or complementary analytical tool for determination of the chemical composition of functionalised surfaces. We emphasise further that with this approach no chemical treatment of IREs is needed; the chips can be regenerated and reused, and applied in other experimental set‐ups.

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