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Protocol 1: Preparation of the HCCA matrix solution in 50% (v/v) aqueous ACN containing 2.5% (v/v) TFA

Protocol 1: Preparation of the HCCA matrix solution in 50% (v/v) aqueous ACN containing 2.5% (v/v) TFA

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2 Sample Preparation



2.3 DIRECT COLONY TRANSFER

The fastest, easiest and cheapest way of obtaining MALDI-TOF mass spectra of intact bacterial cells is the direct transfer of biomass from agar plates or centrifugal

pellets of cultures in liquid media to spots of the stainless steel target and covering

by a matrix layer (Protocol 2a).



Protocol 2a: Direct transfer of bacterial biomass to stainless steel targets

1. Transfer a small, barely visible amount of biomass directly to the sample spot of a

stainless steel MALDI-TOF MS target. A thin film should be obtained by

spreading the cells evenly on the whole area of the spot by using a sterile plastic

pipette tip or inoculation loop. To become acquainted with using the appropriate

amount of cells, please refer to Figure 3.



FIGURE 3

Evaluation of the correct amount of biological material for direct application (Protocol 2a).

(A) MALDI-TOF MS target after application of different amounts of biological material and before

matrix application. Row A, appropriate amount of biological material; row B, lower but still

sufficient amount of biological material; row C, too much biological material. Green (suitable

amount) and red (too high amount) frames mark the same target positions which are shown in

panel (B) after matrix addition and visualised in panel (C) after crystal formation. (B) The same

MALDI target as shown in panel (A) after matrix application and drying. (C) Crystal formation as

observed by the “Camera Image View” of the software package flexControl (Bruker Daltonics):

spots A5 and B4 demonstrate optimal crystal formation; C4 is an example for suboptimal crystal

formation due to excessive biological material applied in the direct transfer step.



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CHAPTER 13 MALDI-TOF MS in Bacterial Systematics



2. Overlay the biological material with 1 ml of the HCCA matrix solution prepared

according to the instructions in Protocol 1.

3. Allow to dry in a fume hood prior to the insertion of the target into the mass

spectrometer.

The quality of spectra may be improved by adding formic acid prior to step 2

(Protocol 2b). The prolonged contact with concentrated acid supports the release

of proteins and gives rise to a better success rate of automated MALDI-TOF

MS measurements for Gram positive bacteria and yeasts. For Gram negative

bacteria, this step is commonly omitted in order to keep the hands-on time per

sample short.



Protocol 2b: Extended direct transfer of bacterial biomass to stainless

steel targets

1. Step 1 of Protocol 2a.

2. Overlay the biomass film with 1 ml of 70% formic acid and allow for drying at

room temperature (RT).

3. Overlay the biomass after formic acid treatment with 1 ml of the HCCA matrix

solution prepared according to the instructions in Protocol 1.

4. Allow to dry in a fume hood prior to the insertion of the target into the mass

spectrometer.



2.4 EXTRACTION METHODS

In early applications of MALDI-TOF MS for bacterial identification, most workers

used direct colony transfer techniques. Only when spectra of insufficient intensity

and resolution, or no spectra at all were obtained, were extraction methods applied

as a last resort. However, recent comparisons of different sample preparation

techniques (Alatoom et al., 2011; Fournier et al., 2012; Saffert et al., 2011;

Schulthess et al., 2013) came unequivocally to the conclusion that methods aimed

at the extraction of proteins prior to the measurement resulted in more high-level

identifications when compared to the direct colony transfer. In a study of 238

strains representing 34 species of anaerobic bacteria, 207 strains were identified

by using the direct colony method while 218 strains were identified after application of an extraction procedure (Fournier et al., 2012). In addition, the score

values indicating the reliability of species identification could be significantly

increased by prior extraction of proteins in this study. Spectra obtained by an

extraction protocol usually show the same diagnostic peaks, less minor peaks of

ambiguous origin and a better S/N ratio than those obtained by direct colony transfer (compare Figure 1A and B). Despite the fact that extraction of proteins requires

additional inconvenient steps for sample preparation, results in a longer turnaround time of analyses and may expose workers to additional chemical

hazards, extraction procedures are on the rise in MALDI-TOF MS identification of bacteria due to their aforementioned advantages (see also Croxatto,

Prod’hom, & Greub, 2012).



2 Sample Preparation



Though proteins can be released by the application of surfactants, enzymatic and

mechanical procedures (Giebel, Fredenberg, & Sandrin, 2008; Meetani & Voorhees,

2005; Teramoto et al., 2007a), ultra sonication (Easterling et al., 1998) and corona

plasma discharge (Birmingham et al., 1999), the extraction of bacterial cells with

solvent systems containing TFA or formic acid turned out to be convenient and to

give rise to high-quality MALDI-TOF mass spectra (Schulthess et al., 2013).

The matrix solvent is the only cell lysing and protein extracting agent when using

direct transfer (Protocol 2a) and it interacts with the biomass only until it has dried

out. Especially for organisms with capsules or a robust cell wall the quality of

MALDI-TOF mass spectra can be significantly improved if proteins are released

from the cells prior to measurements. During the short steps of extraction protocols

several additional factors contribute to the improvement of MALDI-TOF MS measurements: Cells are washed and agar media components like salt, carbohydrates or

peptides are removed. In addition, more biological material can be used and concentrated acids and ACN can be applied for destruction of cell walls and protein release.

The interaction time with the solvents can be carefully controlled, too. Finally a

homogeneous cell-free extract results for application to the target.

Among the solvent-based extraction protocols, the so-called “ethanol-formic acid

extraction” has found the most widespread application (Protocol 3). After step 4 of

Protocol 3, inactivated samples can be shipped at ambient temperature between laboratories or be stored for months at À20  C before finalising the sample preparation

and subsequent measurement.



Protocol 3: Sample preparation by ethanol–formic acid extraction for

MALDI-TOF MS-based identification of bacteria

1. Pipette 300 ml deionised water into an Eppendorf tube.

2. Place biological material (from one single colony up to 5–10 mg) into the tube.

3. Mix thoroughly. Pipetting or vortexing should be appropriate. For

microorganisms which are difficult to suspend, a micro-pistil (Eppendorf 0030

120.973) can be used.

4. Add 900 ml absolute ethanol and mix thoroughly.

5. Centrifuge at approx. 15.800 Â g for 2 min, decant the supernatant, centrifuge

again and remove all the residual ethanol by carefully pipetting it off to waste

without disturbing the pellet.

6. Dry the ethanol-pellet for several minutes at RT.

7. Add 70% formic acid (1–80 ml) to the pellet and mix very well by pipetting

and/or by vortexing.

• If only a little material (one single colony) was used, the volume of formic

acid must be adjusted accordingly (refer to Table 1).

8. Add pure ACN (1–80 ml) and mix carefully.

• Likewise, if only little material (one single colony) was used, the volume of

ACN must be adjusted as well (refer to Table 1).

9. Centrifuge at approx. 15.800 Â g for 2 min until all the material is collected

neatly in a pellet.



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CHAPTER 13 MALDI-TOF MS in Bacterial Systematics



Table 1 Estimates of Volumes of Formic Acid and Acetonitrile used in Protocol

3 Depending on the Amount of Biomass



Formic acid 70%

Acetonitrile



Small Colony



Big Colony



1 ml Loop



10 ml Loop



1–5 ml

1–5 ml



5–15 ml

5–15 ml



10–40 ml

10–40 ml



30–80 ml

30–80 ml



10. Pipette 1 ml of supernatant from the previous step onto a target plate and allow

it to dry in air at normal RT.

• It is recommended to work rapidly in order to avoid oxidation and acylation

of the protein extract which may give rise to artefact peaks.

11. Overlay with 1 ml of matrix solution and allow it to dry in air (in a fume hood)

at ambient temperature.

• It is not recommended to speed up the drying process in steps 10 and 11 by

heating.



2.5 ORGANISM-SPECIFIC SAMPLE PREPARATION

Though Protocol 3 is applicable to sample preparation from the vast majority of

bacteria and allows spectra of high quality to be recorded, certain groups of organisms may cause difficulties in obtaining spectra of sufficient intensities. The experience of users and manufacturers of MALDI-TOF MS identification systems have

resulted in suggestions for optimisation of the standard protocol for specific groups

of bacteria.

Potentially harmful bacteria, in particular, those affiliated to biological safety

level 3 (BSL-3) and classified as group A or group B bioterrorism agents, need to

be inactivated prior to handling outside BSL-3 facilities. Suitable methods for efficient inactivation while maintaining the capability for successful MALDI-TOF

MS-based identification have been reviewed previously (Drevinek et al., 2012;

Dridi & Drancourt, 2011). Protocol 3 was found to be appropriate for inactivation

of viable cells but not of resistant spores. In order to remove spores from the extracts,

the inclusion of centrifugal filtration steps (pore size 0.1 mm) was suggested

(Drevinek et al., 2012; Dybwad, van der Laaken, Blatny, & Paauw, 2013).

A procedure starting with suspending the biomass in 80% (v/v) TFA was suggested

as an alternative to Protocol 3 for reliable inactivation of spores and highly pathogenic bacteria by Freiwald and Sauer (2009) and Lasch et al. (2008).

Many filamentous actinomycetes may cause problems due to the strong aggregation of mycelia and adherence to the agar surface. Cultivation on cellulose nitrate

membrane filters (pore size 45 mm) laid on the agar medium and grinding of the

mycelium scraped from the filters with microtube pestles in water prior to standardised sample preparation allowed successful analyses of streptomycetes and related

organisms (Schumann & Pukall, 2013). Cultivation of filamentous organisms in

liquid media is an alternative remedy for this problem and results additionally



2 Sample Preparation



in a more homogeneous biomass. Experience suggests that mycelial biomass may

strongly absorb aqueous ethanol and requires more intensive drying at step 6 of

Protocol 3, i.e., by application of centrifugal evaporators.

In several cases where bacteria tend to give poor spectra, it has been found

to be advantageous to wash the biomass intensively with deionised water and

aqueous ethanol prior to step 5 of Protocol 3 in order to get rid of salts and other

polar substances that might affect the mass spectrometric ionisation process. It is

suggested to suspend 10–15 mg biomass in 1 ml water by agitation on a vortex

mixer for 2 min. After centrifugation for 2 min, the pellet should be subjected

to the same washing procedure again. The pellet obtained by spinning down for

2 min is carefully re-suspended in 100 ml of water before adding 200 ml of water

and 900 ml of ethanol and mixing with a vortex mixer. Also this washing step

needs to be repeated before the sample can be processed by Protocol 3, starting

at step 5.

Actinomycetes containing mycolic acids (e.g. members of the families Mycobacteriaceae and Nocardiaceae) tend to give only poor spectra possibly due to

their thick lipid cell envelope layers. Additional remedial measures, such as water

washing, mechanical disintegration and heat treatment for inactivation of pathogens, supplementing ethanol–formic acid extraction render the recording of mass

spectra of mycobacteria possible (Protocol 4). Whether or not Protocol 4 is superior

to the usual ethanol–formic acid extraction (Protocol 3) for other mycolata

(e.g. mycolic-acid containing corynebacteria and members of the genera Dietzia,

Gordonia, Millisia, Nocardia, Rhodococcus, Segniliparus, Skermania, Smaragdicoccus, Tomitella, Tsukamurella, Williamsia) and is a remedy for unsatisfactory

results obtained by Protocol 3 needs to be tested in each individual case. Application

of the bead preparation protocol appears to be unnecessary for Nocardia strains and

did not improve the quality of their spectra when compared to the results obtained by

Protocol 3 according to the experience of the authors.



Protocol 4: Bead preparation protocol for MALDI-TOF MS sample

preparation of mycobacteria

A1—Solid medium samples

1. Pipette 300 ml deionised water (or HPLC or MS grade water) into a 1.5 ml

Eppendorf tube.

2. Transfer mycobacteria biomass into the tube. (Avoid collecting medium! Try to

get one to three 10 ml inoculation loops of biomass. As estimate of the amount of

biomass: 2 ml water in an Eppendorf tube represents a small pellet, 5 ml of water

represents a pellet of optimal size).

3. Heat by boiling for 30 min in a water bath (95  C or better 99  C in a thermomixer

may also be suitable).

Warning: The lids of reaction tubes may burst during heat inactivation. Take

precautions to avoid burn injury. This applies to all heat inactivation steps of this

protocol.



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A2—Liquid medium samples, e.g., MGIT™ medium

1. Using a disposable Pasteur pipette, collect 1.2 ml liquid medium from the bottom

of the cultivation tube (where biomass has deposited or will settle within

5–10 min) in a 1.5 ml Eppendorf tube. Centrifuge for 2 min at

15,800–20,000 Â g, then remove medium carefully by pipetting.

2. Add 300 ml deionised water.

3. Heat by boiling for 30 min in a water bath or by using a thermomixer.

B—Follow-up for both solid and liquid medium samples

1. Add 900 ml ethanol and mix thoroughly.

2. Centrifuge for 2 min at 15,800–20,000 Â g and decant supernatant, centrifuge

again and remove residual 75% (v/v) ethanol carefully by pipetting.

3. Allow the pellet to dry at RT for a few minutes.

4. Add beads (0.5 mm Zirconia/Silica beads, BioSpec Products, Catalogue no.

11079105z or similar). Quantity: volume of approximately 100 ml.

5. Add pure ACN (in general 10–50 ml, depending on size of the pellet; if unsure

use 20 ml).

6. Vortex mix at maximum speed for 1 min.

7. Add 70% formic acid (same volume as ACN) and mix by vortexing for approx.

5 s.

8. Centrifuge at 15,800–20,000 Â g for 2 min.

9. Place 1 ml of supernatant on a MALDI target plate and allow to dry at RT.

10. Overlay with 1 ml of HCCA solution immediately after drying.



2.6 MALDI-TOF MS-BASED IDENTIFICATION OF BACTERIA

IN COMPLEX BIOLOGICAL MATRICES

2.6.1 Identification of bacteria in positive blood cultures

Bloodstream infections are associated with high morbidity and mortality (Kumar

et al., 2006). For septic patients, an immediate start of intravenous antibiotics has

great priority. Blood cultures (BC) are one important diagnostic tool. However,

not only timely administration but also the appropriate choice of antimicrobial medication determines the survival rates of patients with septic shock. To speed up the

diagnostic process for bacteraemia, several protocols have been established for

the direct identification of bacteria from positive BC bottles by MALDI-TOF MS.

However, blood and media components interfere with many rapid testing methods,

requiring BC to be processed in order to separate the microorganisms (which represent the minority in terms of biomass in BC) from interfering substances.



Protocol 5: Application of the Bruker MALDI Sepsityper Kit

(Schubert et al., 2011)

1. Transfer 1 ml culture fluids drawn from positive BC bottles to a 1.5-ml

Eppendorf tube.

2. Add 200 ml of the Sepsityper lysis buffer and mix the sample using a vortex mixer

for 10 s.



2 Sample Preparation



3. Centrifuge for 1 min at 17,900 Â g using a centrifuge (e.g. Eppendorf 5417,

Germany).

4. Discard supernatant.

5. Resuspend the pellet in 1 ml of Sepsityper washing buffer.

6. Centrifuge again for 1 min at 17,900 Â g.

7. Discard supernatant.

8. Suspend the pellet in 300 ml of distilled water and continue according

to Protocol 3.



Protocol 6: Differential centrifugation—"Vacutainer" (Gray, Thomas, Olma,

Iredell, & Chen, 2013; Moussaoui et al., 2010)

1. Recover approx. 1.5 ml from positive BC vials and inject it with a syringe into a

gel separator tube (e.g. Clot Activator and Gel BD Vacutainer tubes [Becton

Dickinson] or ZSerumSeptClotActivator [Greiner Bio One, Courtaboeuf,

France]).

2. Centrifuge the tubes at 500 Â g for 10 min at RT to separate blood cells at the

bottom of the gel from plasma, cell debris and bacteria at the surface of the gel.

3. Suspend the surface bacterial sediment in 1.5 ml of sterile water and transfer it to

an Eppendorf tube.

4. Centrifuge at 300 Â g for 1 min at RT for complete removal of cell debris still

present.

5. Transfer 1 ml of the supernatant to another microtube to collect bacteria after a

10,000 Â g centrifugation for 2 min at RT.

6. Suspend the pellet in 300 ml of distilled water and continue according to Protocol 3.



Protocol 7: Processing of positive BC containing charcoal

(Wüppenhorst et al., 2012)

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.



1



Collect 5 ml of positive BC broth.

Centrifuge 10 min at 400 Â g (charcoal and blood cells are removed).

Transfer 1 ml of the supernatant to another microtube.

Add 200 ml 5% saponin1 for cell lysis (Sigma-Aldrich, St. Louis, MO, USA).

Incubate the mixture for 5 min at RT.

Remove remaining charcoal and blood cells with a centrifugation step using a

SigmaPrep™ spin column (800 ml, Sigma-Aldrich, St. Louis, MO, USA).

Centrifuge for 2 min at 330 Â g, collecting the filtrate.

Centrifuge the filtrate for 2 min at 15,800 Â g.

Remove supernatant carefully and wash the pellet in 1 ml of ultra-pure water.

Centrifuge again for 1 min at 15,800 Â g.

Discard the supernatant.

Suspend the pellet in 300 ml of distilled water and continue according to

Protocol 3.



Several authors report on the use of alternative detergents (Ferroni et al., 2010; Meex et al., 2012;

Saffert et al., 2011).



287



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