MALDI-TOF MS: applications to the clinical laboratory 2013년 대한임상미생물학회 5월 집담회 2013.05.23 MALDI-TOF MS: applications to the clinical laboratory PARK, Kyoung Un M.D. Laboratory Medicine, Seoul National Univ. College of Medicine Laboratory Medicine, Seoul National Univ. Bundang Hospital

Today’s contents ▌ Introduction to MALDI-TOF MS ▌ Introduction to SNUBH experiences ▌ MALDI-TOF MS vs. PCR/ESI-MS ▌ MALDI-TOF MS without subculture ▌ Three MALDI-TOF MS systems ▌ Detection of resistance determinants using MALDI ▌ MALDI for HBV DR, HPV genotyping, and so on

Introduction to MALDI-TOF MS

MALDI in microbiological diagnostics (Appl Microbiol Biotechnol 2012) ● Analytes Red spheres Light blue spheres Orange spheres ● Matrix Green spheres ▌ Principle of MALDI-TOF MS identification of bacteria and yeast (spectra = peaks based on mass/charge ratio) ● Laser impact causes thermal desorption of (ribosomal) proteins of bacteria/yeast embedded in matrix material. ● In an electric field, ions are accelerated according to their mass and electric charge. ● The drift path allows further separation and leads to measurable differences in time of flight of the desorbed particles. ● From the time of flight, the exact mass of the polypeptides can be calculated.

MALDI in microbiological diagnostics (Appl Microbiol Biotechnol 2012) ▌ Procedural overview ● Subcultured colonies are applied on a clean target (a). ● Samples are overlaid with matrix solution and air dried (b). ● Measurement in the MALDI-TOF MS is conducted (c). ● An analyte-specific mass spectrum is obtained (d). ● Analyte identification through matching of the generated mass spectrum with spectra in the DB (e).

New technologies in clinical microbiology (Camp Clin Micro 2011) ▌ MALDI-TOF MS ● The protein biomarkers that are measured in whole-microorganism MS - Highly expressed proteins responsible for housekeeping functions (such as ribosomal, chaperone, and transcription/translation factor proteins) ● MALDI relies on ID of protein profiles derived from highly conserved proteins. - Protein profiles: generated from direct ionization of an intact colony - Protein profiles: also generated from a bacterial protein extract after manual extraction ● ID occurs after a protein’s spectral signature is correlated to a DB of spectra. - MALDI in its current form requires subculture prior to identification. - MALDI does not require batching or a prefractionation step. - MALDI generally measures all m/z between 2 and 20 kDa. ● Typical MALDI experiment - Colony selection, placement on a target, addition of matrix, and analysis with MALDI

MALDI in microbiological diagnostics (Appl Microbiol Biotechnol 2012) ▌ MALDI-TOF mass spectrum of Enterococcus faecium ● The measured range of 3,000 to 11,000 Da is displayed. ● The characteristic mass peaks are predominantly ribosomal proteins. ● Subsequently, the integrated MALDI software matches the pattern with entries of a database.

MALDI in microbiological diagnostics (Appl Microbiol Biotechnol 2012) ▌ Culturing organisms & streaking on target plate without or with extraction ● Currently used MALDI-TOF MS techniques are nearly independent of culture conditions. ● Little sample preparation other than streaking a colony on the target plate Directly streaked colonies used for analysis should be as fresh as possible (not more than 48 h). Because with increasing cultivation time, weaker and less distinguished peaks will appear in the spectra. (Probably due to ribosomal protein degradation) ● Extraction for samples that may have strong cell walls (e.g. yeast) The colony to be analyzed is suspended in an 80% ethanol solution. It is centrifuged and resuspended in an acetonitrile and formic acid solution. The resulting supernatant is again centrifuged and placed on the target plate for analysis. * Quick: directly on the target plate by adding 1 μL of neat formic acid on the dried crude sample spot

Mass spectrometry for microbial proteomics ● Whole cell MALDI-TOF mass spectra of P. mirabilis DSM 4479 acquired after the cultivation times indicated. The strain was grown on Columbia blood agar at 37°C and shows a remarkably stable profile in spite of gross changes taking place within the cell during various phases of growth.

Mass spectrometry for microbial proteomics ● MALDI spectra (m/z 2,000-14,000) of Y. enterocolitica 51871 with different growth media Limited tests with the automated peak extraction and library matching showed that ID of bacteria was correct based on the common biomarker signals and despite the overall spectrum variations, although growth conditions and culture time were the major cause of spectrum variation. (M9: minimal medium)

MALDI in microbiological diagnostics (Appl Microbiol Biotechnol 2012) ▌ ID results after comparison of the generated spectrum with the MALDI-TOF database ● The ten best matching entries are shown in a tabular form. ● The degree of similarity to the reference spectrum is represented by a score value. ● ID results with score values above 2.0 are considered to be correct for determination of the respective species.

Two MALDI systems for NFGNB (JCM 2012) ▌ Bruker Biotyper MALDI & VITEK MS MALDI for identification of NFGNB ● Two-hundred NFGNB recovered from cystic fibrosis patient cultures ● Bruker Biotyper (software v3.0) & VITEK MS (SARAMIS database v3.62) ● Bruker Biotyper vs. biochemical or molecular method Agreed for 72.5% of isolates to the species level Agreed for 5.5% to complex / Agreed for 19% to genus 3% not identified ● VITEK MS vs. biochemical or molecular method Agreed for 80% of isolates to the species level Agreed for 3.5% to complex / Agreed for 6% to genus / Agreed for 3.5% to family 7% not identified ● Agreement of combined species/complex/genus level ID with the reference method Higher for Bruker Biotyper (97% vs. 89.5%, P=0.004) (But requiring an extraction step more often) ● Species level agreement with the reference method Similar for both MS systems (72.5% and 80%, P=0.099)

Two MALDI systems for NFGNB (JCM 2012) ▌ Bruker Biotyper MALDI & VITEK MS MALDI ● Subculture on 5% sheep blood agar and incubation for 24 h at 35°C in 5-10% CO2 ● MALDI analysis within 6 h after removal from the incubator ● A thin film of fresh colony growth was applied directly to MALDI-TOF target plates. ● One spot inoculation on the target plate for Bruker & two spots for VITEK MS ● Each spot was then covered with matrix solution, allowed to dry and analyzed. ● Extraction procedure Only if two attempts at direct colony testing failed to provide an acceptable MALDI ID Bacteria were suspended in 70% ETOH followed by centrifugation for 2 min. Supernatant removal The pellet was dried and resuspended in 50 μL 70% formic acid and 50 μL acetonitrile. Centrifugation for 2 min 1 μL supernatant was applied to a spot (2 spots if VITEK MS) on the MALDI-TOF plate and dried. 1 μL matrix was added, dried, and the target plate was placed in the instrument for analysis.

Two MALDI systems for NFGNB (JCM 2012) ▌ Bruker Biotyper MALDI ● Manufacturer recommended score cutoffs Genus level ID: 1.7000-1.999 Species level ID: ≥2.000 ● Additional criteria for different species or genus First ID score has to be at least 10% different from the 2nd ID score to be accepted. ▌ VITEK MS MALDI ● ID were considered acceptable based on the following confidence scores for each spot. One spot result >90% confidence with no conflicting result >85% OR One spot result >85% confidence with other spot corroborating result

Difficult-to-identify bacterial strains (JCM 2011) ▌ ID yield of MALDI-TOF MS analysis for 410 bacterial isolates identified at the species level by 16S SQ MALDI identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species.

Current limitations of MALDI (Camp Clin Micro 2011) ▌ Current limitations associated with MALDI-TOF MS ● The ability to resolve mixtures is lacking. ● A high number of bacterial cells are required for identification. (such that a whole intact colony is typically used for analysis) (limiting the ability to rapidly identify microorganisms directly from biological fluids) ● Requirement of parallel culture-based recovery of positive blood cultures for AST