Department of Plant Science

1
Use of Intact Cell MALDI Fingerprints to Identify
Bacillus and Pseudomonas Isolates

• Department of Plant Science
• National University of Ireland Cork

2
Introduction

• Identification of bacteria by traditional
  molecular and biochemical methods is relatively
  slow and time consuming (and, in the case of
  molecular techniques, expensive).
• An alternative to these is chemotaxonomy the
  identification of bacteria by protein markers.
  Mass spectrometry is a powerful tool for such
  identification.
• Classic mass spectrometry has an upper limit of
  1000 Daltons. Compounds larger than this fracture
  under the laser power used to ionise the sample.
• Matrix Assisted Laser Desorption/Ionisation Tine
  of Flight (MALDI ToF) mass spectromterry
  overcomes this problem by using a matrix to
  buffer the laser power, thus reducing unwanted
  fragmentation.

3
Principals of MALDI ToF Mass Spectrometry

• Matrix Assisted Laser Desorption/Ionisation Time
  of Flight mass spectrometry (MALDI ToF MS) was
  developed in 1987 by Hillenkamp and Karas and
  Tanaka.
• This technique uses a biomolecule embedded in a
  co-precipitate of an UV-light absorbing matrix
  which is irradiated by a nanosecond laser pulse.

4
Principals of MALDI ToF Mass Spectrometry

• Most of the laser energy is absorbed by the
  matrix, which prevents unwanted fragmentation of
  the biomolecule.
• The ionized biomolecules are accelerated in an
  electric field and enter the flight tube.
• During the flight in this tube different
  molecules are separated according to their mass
  to charge ratio and reach the detector at
  different times. In this way each molecule yields
  a distinct signal.

5
Principals of MALDI ToF Mass Spectrometry
Bacterial Analysis

• MALDI analysis of bacterial isolates uses unique
  components, or biomarkers, to differentiate at
  the species level.
• Early work focused on phospholipids and other
  small molecules as principal biomarkers.
• Using proteins as biomarkers, MALDI analysis
  allows the use of crude cellular fractions or
  cellular suspensions and the resulting data from
  such complex mixtures provides evidence for
  identification.

6
Principals of MALDI ToF Mass Spectrometry
Bacterial Analysis

• In terms of speed, using whole bacterial
  eliminates all but the simplest sample
  preparation.
• The spectral patterns obtained from this whole
  cell analysis gives characteristic fingerprints,
  which are thought to be from the desorbed
  components of the cell envelope.
• The resulting fingerprints are then entered into
  a data base which allows unknown isolates to be
  cross referenced.