with applications in the control of quality water and the diagnosis of
infectious diseases, as well as in the detection of pathogenic bacterial strains. In such analysis, instruments such as matrix-assisted
laser desorption/ionization time of flight mass spectrometry
(MALDI-TOF MS) and liquid chromatography–electrospray
ionization-tandem mass spectrometry (LC–ESI-MS/MS) [9, 10]
are mainly used. MALDI-TOF-MS has been commonly employed
for the identification of peptidic pattern “fingerprint” specific of
each microorganism [11, 12]. In addition, LC–ESI-MS/MS techniques have been employed for the detection and identification of
bacteria, based on specific diagnostic peptides [9, 13]. These LC–
ESI-MS/MS techniques have proved to be effective also in the
detection of toxin peptides of specific species such as Shiga toxin
for Escherichia coli [14], botulinic toxins [15], and enterotoxins for
Staphylococcus aureus [16]. Also, they have been useful to differentiate between Bacillus cereus group species, based on their peptide
profile comparison [10]. Additionally, other peptide profiles, analyzed using LC–MS/MS [13], have been published, for new diagnosis peptide discoveries, such as S. aureus mastitis producer strains
peptides.
These mass spectrometry techniques used for the identification
and determination of specific species or strain diagnosis peptides are
fast, require few materials, and also are specific, sensitive, and
reproducible for the identification of pathogenic bacteria. In addition, there are endless possibilities on the number of samples that
can be analyzed.
2 Materials
2.1 Strain Culture
1. Brain heart infusion medium (BHI).
2. Plate count agar medium (PCA).
3. Incubator set at 31
C.
2.2 Protein
Extraction
1. 50% acetonitrile (50% ACN) and 1% aqueous trifluoroacetic
acid (1% TFA).
2. 75 mM NaCl and 50 mM Tris–HCl.
3. 80% TFA.
4. Dismembrator.
5. Centrifuge.
6. Lauryl maltoside buffer lysis solutions: 60 mM Tris–HCl
pH 7.5, 1% lauryl maltoside, 5 mM phenylmethanesulfonyl
fluoride (PMSF), and 1% dithiothreitol (DTT).
7. n-dodecyl-D-maltoside buffer lysis solution: 2% of 75 mM ndodecyl-D-maltoside NaCl and 50 mM Tris–HCl.
8. Speedvac.
206
Ana G. Abril et al.
infectious diseases, as well as in the detection of pathogenic bacterial strains. In such analysis, instruments such as matrix-assisted
laser desorption/ionization time of flight mass spectrometry
(MALDI-TOF MS) and liquid chromatography–electrospray
ionization-tandem mass spectrometry (LC–ESI-MS/MS) [9, 10]
are mainly used. MALDI-TOF-MS has been commonly employed
for the identification of peptidic pattern “fingerprint” specific of
each microorganism [11, 12]. In addition, LC–ESI-MS/MS techniques have been employed for the detection and identification of
bacteria, based on specific diagnostic peptides [9, 13]. These LC–
ESI-MS/MS techniques have proved to be effective also in the
detection of toxin peptides of specific species such as Shiga toxin
for Escherichia coli [14], botulinic toxins [15], and enterotoxins for
Staphylococcus aureus [16]. Also, they have been useful to differentiate between Bacillus cereus group species, based on their peptide
profile comparison [10]. Additionally, other peptide profiles, analyzed using LC–MS/MS [13], have been published, for new diagnosis peptide discoveries, such as S. aureus mastitis producer strains
peptides.
These mass spectrometry techniques used for the identification
and determination of specific species or strain diagnosis peptides are
fast, require few materials, and also are specific, sensitive, and
reproducible for the identification of pathogenic bacteria. In addition, there are endless possibilities on the number of samples that
can be analyzed.
2 Materials
2.1 Strain Culture
1. Brain heart infusion medium (BHI).
2. Plate count agar medium (PCA).
3. Incubator set at 31
C.
2.2 Protein
Extraction
1. 50% acetonitrile (50% ACN) and 1% aqueous trifluoroacetic
acid (1% TFA).
2. 75 mM NaCl and 50 mM Tris–HCl.
3. 80% TFA.
4. Dismembrator.
5. Centrifuge.
6. Lauryl maltoside buffer lysis solutions: 60 mM Tris–HCl
pH 7.5, 1% lauryl maltoside, 5 mM phenylmethanesulfonyl
fluoride (PMSF), and 1% dithiothreitol (DTT).
7. n-dodecyl-D-maltoside buffer lysis solution: 2% of 75 mM ndodecyl-D-maltoside NaCl and 50 mM Tris–HCl.
8. Speedvac.
206
Ana G. Abril et al.