13. During trypsinization, the pH is 7. After the addition of 60 μl
of FA 5%, the pH is revised by reactive strips. In case, the
protocol is not finished, the samples may be stocking at
À20
C.
14. This last step is repeated until the entire volume of the sample is
loaded onto the column. Normally, 3 loadings of 200 μl, discarding the eluate.
15. In case the mass spectrometry analysis is not performed, the
samples may be stocked at À80
C.
16. Different LC–MS instruments from different commercial
brands may use specific parameters. Another example is as
Carrera et al. [13] analyzed protein extraction by LC–MS/
MS using a Proxeon EASY-nLC II Nanoflow system (Thermo
Fisher Scientific, San Jose, CA) coupled to a LTQ-Orbitrap XL
mass spectrometer (Thermo Fisher Scientific). In this case, the
used search engine is SEQUEST-HT (Proteome Discoverer
2.1, Thermo Fisher Scientific).
17. For a more accurate searching, run generated data against
genera or larger taxonomic group that the analyzed strains
belong to.
18. For the BLASTp search, bacterial species or genus of each
sample is included and excluded with the aim of finding
which peptides belonged exclusively to the analyzed strain.
References
1. Chakravorty S, Helb D, Burday M et al (2007)
A detailed analysis of 16S ribosomal RNA gene
segments for the diagnosis of pathogenic bacteria. J Microbiol Methods 69:330–339
2. Ivnitski D, Abdel-hamid I, Atanasov P et al
(1999) Biosensors for detection of pathogenic
bacteria. Biosens Bioelectron 14:599–624
3. Bo ¨hme K, Ferna ´ndez-No IC, Barros-Vela ´zquez J et al (2011) Rapid species identification
of seafood spoilage and pathogenic grampositive bacteria by MALDI-TOF mass fingerprinting. Electrophoresis 32:2951–2965
4. Bo ¨hme K, Morandi S, Cremonesi P et al
(2012) Characterization of Staphylococcus
aureus strains isolated from Italian dairy products by MALDI-TOF mass fingerprinting.
Electrophoresis 33(15):2355–2364
5. Branquinho R, Sousa R, Lopes C et al (2014)
Differentiation of Bacillus pumilus and bacillus
safensis using MALDI-TOF-MS. PLoS One 9
(10):e110127
6. Lasch P, Beyer W, Nattermann H et al (2009)
Identification of Bacillus anthracis by using
matrix-assisted laser desorption ionizationtime of flight mass spectrometry and artificial
neural networks. Appl Environ Microbiol 75
(22):7229–7242
7. Quintela-Baluja M, Bo ¨hme K, Ferna ´ndez-No
IC et al (2014) MALDI-TOF mass spectrometry, rapid and reliable method for the identification of bacterial species in food microbiology
laboratories. In: Boziaris IS (ed) Novel food
preservation and microbial assessment techniques. CRC Press, Boca Raton, Florida
8. Doeun D, Davaatseren M, Chung MS (2017)
Biogenic amines in foods. Food Sci Biotechnol
26(6):1463–1474
9. Calo-Mata P, Carrera M, Bo ¨hme K et al (2016)
Novel peptide biomarker discovery for detection and identification of bacterial pathogens
by LC-ESI-MS/MS. J Anal Bioanal Tech
7:1):1–1):9
10. Pfrunder S, Grossmann J, Hunziker P,
Brunisholz R, Gekenidis MT, Drissner D
(2016) Bacillus cereus group-type strainspecific
diagnostic peptides. J Proteome Res 15
(9):3098–3107
212
Ana G. Abril et al.
of FA 5%, the pH is revised by reactive strips. In case, the
protocol is not finished, the samples may be stocking at
À20
C.
14. This last step is repeated until the entire volume of the sample is
loaded onto the column. Normally, 3 loadings of 200 μl, discarding the eluate.
15. In case the mass spectrometry analysis is not performed, the
samples may be stocked at À80
C.
16. Different LC–MS instruments from different commercial
brands may use specific parameters. Another example is as
Carrera et al. [13] analyzed protein extraction by LC–MS/
MS using a Proxeon EASY-nLC II Nanoflow system (Thermo
Fisher Scientific, San Jose, CA) coupled to a LTQ-Orbitrap XL
mass spectrometer (Thermo Fisher Scientific). In this case, the
used search engine is SEQUEST-HT (Proteome Discoverer
2.1, Thermo Fisher Scientific).
17. For a more accurate searching, run generated data against
genera or larger taxonomic group that the analyzed strains
belong to.
18. For the BLASTp search, bacterial species or genus of each
sample is included and excluded with the aim of finding
which peptides belonged exclusively to the analyzed strain.
References
1. Chakravorty S, Helb D, Burday M et al (2007)
A detailed analysis of 16S ribosomal RNA gene
segments for the diagnosis of pathogenic bacteria. J Microbiol Methods 69:330–339
2. Ivnitski D, Abdel-hamid I, Atanasov P et al
(1999) Biosensors for detection of pathogenic
bacteria. Biosens Bioelectron 14:599–624
3. Bo ¨hme K, Ferna ´ndez-No IC, Barros-Vela ´zquez J et al (2011) Rapid species identification
of seafood spoilage and pathogenic grampositive bacteria by MALDI-TOF mass fingerprinting. Electrophoresis 32:2951–2965
4. Bo ¨hme K, Morandi S, Cremonesi P et al
(2012) Characterization of Staphylococcus
aureus strains isolated from Italian dairy products by MALDI-TOF mass fingerprinting.
Electrophoresis 33(15):2355–2364
5. Branquinho R, Sousa R, Lopes C et al (2014)
Differentiation of Bacillus pumilus and bacillus
safensis using MALDI-TOF-MS. PLoS One 9
(10):e110127
6. Lasch P, Beyer W, Nattermann H et al (2009)
Identification of Bacillus anthracis by using
matrix-assisted laser desorption ionizationtime of flight mass spectrometry and artificial
neural networks. Appl Environ Microbiol 75
(22):7229–7242
7. Quintela-Baluja M, Bo ¨hme K, Ferna ´ndez-No
IC et al (2014) MALDI-TOF mass spectrometry, rapid and reliable method for the identification of bacterial species in food microbiology
laboratories. In: Boziaris IS (ed) Novel food
preservation and microbial assessment techniques. CRC Press, Boca Raton, Florida
8. Doeun D, Davaatseren M, Chung MS (2017)
Biogenic amines in foods. Food Sci Biotechnol
26(6):1463–1474
9. Calo-Mata P, Carrera M, Bo ¨hme K et al (2016)
Novel peptide biomarker discovery for detection and identification of bacterial pathogens
by LC-ESI-MS/MS. J Anal Bioanal Tech
7:1):1–1):9
10. Pfrunder S, Grossmann J, Hunziker P,
Brunisholz R, Gekenidis MT, Drissner D
(2016) Bacillus cereus group-type strainspecific
diagnostic peptides. J Proteome Res 15
(9):3098–3107
212
Ana G. Abril et al.