MALDI-TOF MS-Based characterization of Microorgamisms: A Microbiology Course Lab Exercise

I. Introduction 

The development of rapid and effective methods of identifying microorganisms is extremely important to many fields including medicine, food safety and microbiology, counter bioterrorism, and environmental monitoring (Schumacher et al. 2012).  Current methods, like PCR, are effective, but involve complex preparation and costly materials (Ng 2013).  The increase in technology and different research has lead to a more rapid and cost effective way.  The method is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which leads to a new effective way of identifying microorganisms (Ng 2013).  It has been proven as a promising tool to profile bacteria at the genus and species level and sub species level (Schumacher et al. 2012).

OBJECTIVES:

1)     To gain familiarity using the MALDI-TOF MS method.

2)     To use FlexAnaylsis to obtain a spectra and peaks of your microorganism.

3)     To use SPECLUST or other software to construct a dendrogram and obtain other statistical data regarding your microorganism. 

II. Background Information 

How MALDI-TOF Works

Spectrometry involves measuring the mass of certain components, in our case proteins, of the sample in question.  The MALDI instrument has three major parts: the ionization source, the analyzer, and the detector.  The sample is first loaded into the instrument on a stainless steel plate.  Once the sample is in the machine, it goes through ionization and the particles become electrically charged. After the sample is in the mass analyzer region of the spectrometer there are a series of shots that are fired from a laser at a controlled power. Once the certain molecules are hit the time is recorded that it takes for them to go through the time-of-flight tube and to the detector.  The mass to charge, signal to noise, amplitude, and intensities of the peaks are all recorded creating a spectra of proteins found in the particular microorganism (Tuma 2003).  

III. Procedures

Sample Preparation

 When it comes to preparing samples for the MALDI there are two common methods: intact cell and protein extraction. Protein extraction has proven to be a method that gives less noise in the spectra and increases reproducibility among other advantages (Bohme et al. 2010). In this method the proteins from the cells are extracted through a series of centrifugations and chemical additions. In the final step of this process the proteins are in a supernatant that is placed in 0.5μl amounts on the MALDI polished stainless steel plate, which is then overlaid with 0.5μl α-cyano matrix. 

 1)     Forming the Pellet

 -Each group will receive their unknown microorganism in a 5ml Nutrient                                 Broth Preparation and a blank nutrient broth sample.     

            a) Turn on the Spectrophotometer to 600A 

            b) Pipette 1ml of your Nutrient Broth Blank into a cuvette and zero the spectrophotometer

            c) Pipette 1ml of your Unknown Nutrient Broth into a cuvette and record OD obtained. (The OD should be around 1.0A, if higher or lower by 0.1A ask your instructor for help.)

            d) Transfer the unknown nutrient broth into a 1.5ml centrifuge tube

            e) Centrifuge your sample for 2 min at 10,000rpm

            f) Pipette off the supernatant and replace with 1ml DD H20

            g) Resuspend the pellet by pipetting 

            h) Centrifuge your sample for 2 min at 10,000 rpm

            i) Pipette off the supernatant and replace with 1ml DD H20        

            j) Resuspend the pellet by pipetting 

            k) Centrifuge your sample for 2 min at 10,000 rpm

            l) Pipette off the supernatant 

2)     Protein Extraction from cells

      a) Suspend the pellet in 300μl of DD H20 and mix by pipetting

      b) Add 900μl of absolute ethanol to your sample and mix by pipetting

      c) Centrifuge the sample for 2 min at 10,000rpm

      d) Pipette off the supernatant 

      e) Centrifuge again for 2 min at 10,000rpm

      f) Pipette off the remaining supernatant 

      g) Allow to air-dry for one  minute

      h) Add 10μl of 70% formic acid to the pellet and mix thoroughly by pipetting

      i) Vortex the sample for 30 seconds

      j) Add 10μl Acetonitrile to the sample and mix thoroughly by pipetting

      k) Vortex the sample for 30 seconds

      l) Centrifuge the sample for 2 min at 10,000rpm

      m) Pipette off the supernatant and add to a new 1.5ml centrifuge tube

3)     Spotting the plate

      a) Pipette 0.5μl of the protein extract onto the sample well and allow to dry.

      b) Repeat step a in three different wells

      c) After the sample has dried, overlay each of your sample wells with 0.5μl of 

      α-cyano matrix 

 4)      Obtain your spectra and collect data

Working with data

Putting data in excel

      a) Copy all of the data from Flex Analysis and paste it into an excel workbook

      b) Delete the columns including the Resolution and S/N from each of the wells      

      c) For each well find 15-25 peaks with the highest intensities and paste them into a new excel worksheet.

      d) Once all peaks are in worksheet email the worksheet to the instructor to be able to collect all of the class data

      e) The instructor will email an excel file with all of the unknown’s peaks and corresponding intensities

SPECLUST data Analysis

            a) With the excel file, arrange columns by descending intensity 

            b) Select 15-25 peaks that show the highest intensity 

            c) Transfer the most intense peaks into a file with the suffix “.txt” (This can usually done very simply with the “Notepad” application) Note: data input in the file should be sorted as a list containing ONLY Peak m/z’s

            d) Data for multiple replicates or multiple unknown microorganisms should be compressed into a “.zip” file

            e) Go to the website http://co.bmc.lu.se/speclust/

            f) Click on “Clustering” and then browse to obtain the “.zip” file from your computer

            g) Click “Cluster” to create a dendrogram

            h) Download and print the dendrogram and attach to the lab papers to turn in

Library Spectra Analysis

Using the attached spectra and 15-25 peak m/z’s match up your unknown spectra with the known spectra that are attached

QUESTIONS:

 1)    What is the purpose of adding the formic acid and acetonitrile?

2)     Based on the known spectra given, what is your unknown?

3)     What does your dendrogram show?   

References

Bohme KI, Fernandez-No C, Barros-Velazquez J, Gallardo JM, Canas B, Calo-Mata P.2010.Comparative analysis of protein extraction methods for the identification of seafood-borne pathogenic and spoilage bacteria by MALDI-TOF mass spectrometry. Anal Methods 2(12):1941–1947

 Ng, W. 2013. Teaching Microbial Identification with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and Bioinformatics Tools. Journal of Microbiology & Biology Education. [Online] 14:1

 R. Alm, P. Johansson, K. Hjernø, C. Emanuelsson, M. Ringnér, and J. Häkkinen.                          Detection and identification of protein isoforms using cluster analysis of MALDI-MS mass spectra.Journal of Proteome Research 5, 785-792 (2006)

 Schumaker, S., Borror, C.M. and Sandrin, T.R.2012. Automating data acquisition affects mass spectrum quality and reproducibility during bacterial profiling using an intact cell sample preparation method with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 26: 243–253. 

 Tuma RS. 2003. MALDI-TOF mass spectrometry: Getting a feel for how it works. Oncology Times[Online]; 25(19):26.