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ESI MS/MS biotypization protocol

ESI PROTOCOL FOR IDENTIFICATION OF MICROORGANISMS BY PROTEIN READING CONCEPT

Introduction

The protein-reading concept using CAF-/CAF+ reagent (chemically activated fragmentation negative/chemically activated fragmentation positive) enables fast, highly accurate, reliable and easy to use identification of microorganisms down to the species level. It is one step derivatization reaction easily incorporated into existing workflows commonly used by biological scientists, as outlined in Figure 1.

SAMPLE PREPARATION (Step 1)
Isolate the proteins from your sample & perform trypsin digestion

DERIVATIZATION (Step 2)
Add CAF-/CAF+ reagent to peptides

SEPARATION & ANALYSIS (Step 3)
Separate tagged peptides by UPLC & analyse by ESI-MS/MS

IDENTIFICATION (Step 4)
Identify microorganism by ESI-MS/MS & ProteinReader*

Figure 1. Workflow of ESI-MS/MS identification of microorganisms using CAF-/CAF+ reagent.

*ProteinReader is expert software developed exclusively for species identification

 

CAF-/CAF+ is a chemical reagent for the derivatization of peptide samples prior to analysis by ESI (Electrospray Ionization) Tandem Mass Spectrometry (MS/MS). Derivatization procedure of tryptic peptides at N-terminus is illustrated by the following reaction (Figure 2.):

reaction with CAF

Figure 2. Derivatization procedure of tryptic peptide with CAF-/CAF+ reagent (5-formylbenzene-1,3-disulfonic acid).

 

For the first time, mass spectrometry can exploit both positive and negative ion mode, either for species or proteins identification.

The CAF-/CAF+ method enables de novo sequencing of derivatized peptides with negative and positive ion mode tandem mass spectrometry (MS/MSand MS/MS+). Peptide sequences are read from MS/MS spectra and matched against the NCBInr database by developed software named ProteinReader and confirmed by the mass spectrometry data of elucidated peptide mass sequences derived from the annotated genome.

Benefits of using CAF-/CAF+ derivatization reagent

Accurate

CAF-/CAF+ is a mild derivatizing reagent with no side reactions resulting in peptide fingerprinting exclusively in negative ion mode (MS) without interferences and adducts (e.g. sodium, potassium, ammonium) and peptide amino acid sequence analysis in positive and negative MS/MS+/-.

Fast

CAF-/CAF+ reagent significantly reduces the derivatization time to only 10 minutes.

The whole process of sample identification from sample preparation to species identification can be finished in maximum 4 h depending on sample complexity.*

Reliable

CAF-/CAF+ reagent allows de novo sequencing from spectra of both b-ions (obtained in negative ion mode) and y-ions (obtained in positive ion mode) making sequence reading unambiguous and easy by using two orthogonal techniques (MS/MS negative and MS/MS positive). By reading the same sequence twice forward and backward probability of misreading or misinterpretation of microorganism ID is minimized.

*The protocol consists of two phases: First is sample preparation (protein extraction and digestion) and second is sample treatment & processing (derivatization, separation and identification). Sample preparation can take from one to two hours depending on sample complexity and digestion method used (trypsin tip or trypsin magnetic beads). n. b. overnight in-solution tryptic digestion (9-18 h) can be applied, as well.

 

Materials, reagents, instruments, software

Materials

Materials enclosed in brackets are the ones used in our laboratory procedures.

  • Tissue homogenizer (TissueRuptor and TissueRuptor disposable probes, Quiagen, Germany)
  • 2.0 mL PP tubes for cell lysis (Eppendorf, Germany)
  • 1.5 mL microcentrifuge tubes (Eppendorf, Germany)
  • Tube holder for 1.5 mL tubes
  • Tube holder cool pack for 1.5 or 2.0 mL tubes
  • Vortexer for mixing
  • Laboratory centrifuge with centrifugal force of at least 2,000 rcf (Centric 400, Tehtnica, Slovenia)
  • Bench-top microcentrifuge with cooling system and centrifugal force of at least 4,500 rcf (Centrifuge 5415 R, Eppendorf, Germany)
  • Vacuum concentrator (Concentrator 5301, Eppendorf, Germany)
  • Thermomixer (Thermomixer comfort, Eppendorf, Germany)
  • Household microwave oven (HeatWave compact 800W, Electrolux)
  • LC Trap Column (ACQUITY UPLC PST C18 nanoACQUITY Trap, 100Å, 5 µm, 180 µm × 20 mm, Waters, USA)
  • LC Analytical Column (ACQUITY UPLC PST C18 nanoACQUITY Column, 130Å, 1.7 µm, 100 µm × 100 mm, Waters, USA)

Reagents used

  • Ammonium bicarbonate, ≥ 99.0% (Sigma-Aldrich)
  • Triton X-100 (Sigma-Aldrich)
  • Mag-Trypsin, trypsin immobilized on magnetic beads (Clontech, USA)
  • Acetonitrile, gradient grade for liquid chromatography (Merck, Germany)
  • Trifluoracetic acid, for spectroscopy (Merck, Germany)
  • Formic acid, 98-100% (Kemika, Croatia)
  • CAF-/CAF+ reagent (5-formylbenzene-1,3-disulfonic acid disodium salt hydrate, p.a. synthetic product, Ruđer Bošković Institute)
  • Sodium cyanoborohydride, for synthesis (Merck, Germany)
  • Potasium dihydrogen phosphate, p.a. (Kemika, Croatia)
  • Ultrapure water, TOC < 5 ppb, resistivity < 18.2 MΩ cm

Instruments

Instruments enclosed in brackets are the ones used in our laboratory procedures.

  • Ultra Performance Liquid Chromatography system (nanoACQUITY UPLC, Waters, USA)
  • ESI-MS/MS mass spectrometer (Synapt G2-Si Mass Spectrometer, Waters, USA)

Software

  • Protein Reader (software developed by “Ruđer Bošković” Institute and Faculty of Food Technology and Biotechnology, University of Zagreb)

Procedure

STEP 1 – SAMPLE PREPARATION

1. Removing cell suspension growth medium

Centrifuge suspension of cells in 50 or 15 mL culturing tubes 2,000 rcf for 15 min and aspirate the supernatant. Resuspend the pellet in 1.5 mL of 25 mM NH4HCO3, transfer to a 2.0 mL tube and centrifuge at 4,500 rcf for 20 min at 4°C. Aspirate the supernatant. Repeat 2-4 more times, depending on the sample.

2. Cell lysis

Add 400 µL of cell lysis buffer (25 mM NH4HCO3 + 0.1% Triton-X-100) and resuspend the pellet. Keep the tubes incubated on cool pack.
Proceed to cell rupture using TissueRuptor and disposable probe with the following settings:
Power: Position II
Grinding cycle: 45 seconds ON / 45 seconds OFF
Total grinding times: 5-6 cycles

3. Blocking endoprotease activity

Stop the TissueRuptor after 5 cycles, close the lid on 2.0 mL tube and put the sample in boiling water for 3 minutes to inhibit endoprotease activity.

4. Obtaining solution of proteins

Remove the sample tube from the water bath and centrifuge at 4,500 rcf for 20 min at 4°C to remove disrupted cell material. Transfer the supernatant containing soluble proteins to a clean 1.5 mL tube.

5. Protein digestion

Use one of the three trypsin digestion procedures:

a. Mag-Trypsin (trypsin immobilized on magnetic beads)

b. Trypsin tip

c. Standard in-solution tryptic digestion

STEP 2 – DERIVATIZATION

6. Derivatization procedure

Reconstitute each sample containing the evaporated tryptic peptide mixture with 60 µL of derivatization solution. The derivatization solution contains 12.5 mM 5-formylbenzene-1,3-disulfonic acid disodium salt hydrate (p.a. synthetic product, Ruđer Bošković Institute) and 95.5 mM of NaBH3CN dissolved in 10 mM KH2PO4 and adjusted to pH 5. Close the tube with the sample, place it in the holder (e.g. styrofoam) and perform the derivatization in a household microwave oven at 180W for 8 minutes.

STEP 3 – SEPARATION

7. Separation by ultra-performance liquid chromatography

The nanoACQUITY UPLC system equipped with a sample manager and two binary solvent managers is used for peptide separation. Perform the multi-step chromatography using trap column C18, 180 µm × 20 mm, 5 µm particle size and analytical column C18, 100 µm × 100 mm, 1.7 µm particle size. Maintain the column temperature at 40°C. Set the flow rate to 1 µL/min and injection volume to 1 μL. Vial temperature is maintained at 10°C in the autosampler tray. Use following mobile phases: mobile phase A consists of 0.1% HCOOH aqueous solution and mobile phase B consists of 0.1% HCOOH in 95% ACN (v/v). Program the 30 min gradient elution to increase the percentage of solvent B from 1% to 99% over 22 min and then to condition the column back to the initial conditions until completion of the run. Set complete gradient conditions following the conditions below:

Binary solvent manager 1

Analytical column – complete gradient conditions

Time (min) Flow (µL/min) A (%) B (%)
initial 1.0 99.0 1.0
0.10 1.0 99.0 1.0
20 1.0 40.0 60.0
22 1.0 1.0 99.0
24 1.0 99.0 1.0

 

Trapping column – conditions

Flow: 1 µL/min                 A1=99.0%, B1=1.0%

 

Binary solvent manager 2

Trapping column – complete gradient conditions

Time (min) Flow (µL/min) A (%) B (%)
initial 10.0 100.0 0.0

 

Analytical column – conditions

Flow: 1 µL/min                 A1=99.0%, B1=1.0%

 

fig3a

fig3b

Figure 3. Example of UPLC chromatograms for derivatized BSA in (A) positive and (B) negative ion mode.

 

The nanoACQUITY UPLC is directly coupled to the mass spectrometer SYNAPT G2-Si. Eluted derivatized peptides are detected by mass spectrometry.

 

STEP 4 – IDENTIFICATION

8. Mass spectrometry

The samples are analyzed by an electrospray LC-MS/MS approach using a data dependant acquisition (DDA) method in both positive and negative mode. Peptide fragmentation is performed in the trap collision cell with argon as the collision gas in ESI+ and ESI- modes.

Accurate mass LC-MS/MS DDA data for positive electrospray (ESI+) mode were obtained as follows. MS survey was obtained in a mass range of m/z 400 to 2000 and MS survey scans of 0.05 s duration with an interscan delay of 0.01 s were acquired. MS/MS data (m/z 50 to 2000) were obtained for up to five ions of charge 2+, 3+, 4+ or 5+ detected in the survey scan. MS/MS was obtained at a scan rate of 0.2 with 0.01 s interscan delay and a collision energy ramp from 16-21 eV (for low mass) to 62-78 eV (for high mass). Acquisition was switched from MS to MS/MS mode when the base peak intensity (BPI) exceeded a threshold of 30.000 counts, and returned to the MS mode when the TIC in the MS/MS channel exceeded 100.000 counts/s or when 1 s were acquired.

Accurate mass LC-MS/MS DDA data for negative electrospray (ESI-) mode were obtained as follows. MS survey was obtained in a mass range of m/z 500 to 3000 and MS survey scans of 0.5 s duration with an interscan delay of 0.01 s were acquired. MS/MS data (m/z 50 to 3000) were obtained for up to ten ions of charge 1- detected in the survey scan. MS/MS was obtained at a scan rate of 0.1 with 0.01 s interscan delay and a collision energy ramp from 30-40 eV (for low mass) to 90-100 eV (for high mass). Acquisition was switched from MS to MS/MS mode when the base peak intensity (BPI) exceeded a threshold of 1000 counts, and returned to the MS mode when the TIC in the MS/MS channel exceeded 100.000 counts/s or when 1 s were acquired.

Nanoflow conditions were set and controlled from the Tune Page:

Positive ion mode (ESI+)

SOURCE

  • Capillary: 4.0 kV
  • Sampling cone: 40
  • Source offset: 40

TEMPERATURE

  • Source: 80 °C

GAS FLOWS

  • Cone gas (L/h): 0
  • Nano Flow Gas (Bar): 0.5
  • Purge Gas (L/h): 0

 

Negative ion mode (ESI-)

SOURCE

  • Capillary: 2.5 kV
  • Sampling cone: 40
  • Source offset: 40

TEMPERATURE

  • Source: 80 °C

GAS FLOWS

  • Cone gas (L/h): 0
  • Nano Flow Gas (Bar): 1.0
  • Purge Gas (L/h): 0