Investigation of spectral reproducibility in direct analysis of bacteria proteins by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Abstract

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) can be used for rapid detection of bacteria proteins in a crude mixture. It can potentially be used as a tool for bacterial identification based on the mass spectral patterns or the appearance of some characteristic mass peaks. However, there are many experimental parameters that can potentially have a strong effect on the observed mass spectra. The objective of this work is to address the mass spectral reproducibility issue. Several experimental parameters that may affect the MALDI spectra are systematically investigated. Results of spectral comparison from two laboratories with different operators and instrumentation are presented. It is demonstrated that minor variations in the sample/matrix preparation procedures for MALDI and in the experimental conditions used for bacterial protein extraction can result in a significant change in the observed spectra, though a number of peaks are conserved in the spectra obtained under different experimental conditions from the same bacterial sample. These conserved peaks may potentially be used as the biomarkers for bacterial identification. It is stressed that this type of investigation on spectral reproducibility should be carried out for different bacterial species in order to identify the mass spectral peaks that are consistently detected regardless of operator and nominal variations in sample preparation approach.

Applications of MALDI‐TOF Mass Spectrometry in Clinical Diagnostic Microbiology

“…DHB and CHCA are usually optimal for the detection of lower mass ions (Hathout et al, 2000;Williams et al, 2003;Ruelle et al, 2004) with a detection up to 10 kDa when the proper solvent is used. Both SA and FA have been shown to be better for the detection of higher mass ions (above 15 kDa) (Madonna et al, 2000;Conway et al, 2001;Ruelle et al, 2004;Vargha et al, 2006) but provide a lower sensitivity than CHCA (Wang et al, 1998;Ruelle et al, 2004).…”

“…Many experimental parameters can have an important effect on the observed mass spectra (Wang et al, 1998). The reproducibility is dependent on the MALDI-TOF instrument, the matrix used, the age of the microorganism, the sample : matrix ratio, the sample concentration, the culture medium and growth conditions (Valentine et al, 2005).…”

“…The reproducibility is dependent on the MALDI-TOF instrument, the matrix used, the age of the microorganism, the sample : matrix ratio, the sample concentration, the culture medium and growth conditions (Valentine et al, 2005). However, several studies have shown that a subset of peaks from genetically identical bacteria was conserved in spectra obtained in different experimental conditions (Wang et al, 1998;Welham et al, 1998). These conserved peaks, among which ribosomal proteins are well represented, explain the feasibility to use MALDI-TOF for bacterial identification even without the standardization of experimental conditions.…”

“…Studies comparing mass spectra of identical microorganisms obtained in different locations but with the same experimental protocols and instruments hardware and software have shown promising results in terms of inter-laboratory reproducibility. For instance, two studies using different bacterial species have demonstrated that more than 60% of the peaks observed in mass spectra were similar in separate laboratories (Wang et al, 1998;Walker et al, 2002). However, a poor inter-laboratory reproducibility of MALDI-TOF MS of intact microorganisms was demonstrated in a study where three independent laboratories using three different commercial instruments performed a MALDI-TOF MS analysis of identical aliquots of E. coli culture prepared and analysed in the same experimental conditions .…”

Rapid identification of stress-related fingerprint from whole bacterial cells of Bifidobacterium lactis using matrix assisted laser desorption/ionization mass spectrometry

“…Thus, the sample preparation strategy adopted for MALDI-TOFMS of whole bacteria is crucial to improve this reproducibility. Indeed, several studies have shown that the obtained bacterial profiles depend on a number of parameters, such as instrumentation, culture media, and bacterial growth [13][14][15][16][17][18].…”