Fig. 1 MALDI-TOF Mass Spectra (Cytochrome c, 25fmol, Matrix: Sinapic Acid)
(A) with no additive (B) with addition of SDS and anion-exchange silica gel

■Background & Features
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool to analyze and identify minute amount of proteins. However, it has been difficult to obtain sufficient ion intensities originated from insoluble proteins or proteins with ionization-suppressing substances. Hirota et al. developed a sensitivity enhancing method for protein MS analysis using granular silica gel which bears quaternary ammonium groups as an additive to the matrix without removing SDS. This technique realized highly sensitive detection of the proteins: the detection limit of cytochrome c was improved from 500fmol to 25fmol. The anion-exchange silica gel improves quality of the spectra of the following MALDI-MS measurements:

(1) SDS-containing protein solutions
(2) Insoluble proteins
(3) Proteins with extremely small amount

■Protocols
Mass spectrometer: MALDI-TOF mass spectrometer equipped with a nitrogen laser (337nm)
(1) SDS-Containing Protein Solution
A. Preparation of a Protein Solution
Adjust the SDS concentration to 0.05-0.2% (w/v) of the protein solution that was dissolved in an appropriate buffer solution (e.g. 50mM Tris-HCl (pH7.5)-100mM NaCl).

B. Preparation of Gel-SA (Sinapic Acid) Suspension
i) Dissolve SA into 50% acetonitrile-0.1% (v/v) TFA to adjust the concentration to 10mg/mL.
ii) Add 50mg of the ion-exchange silica gel to this 1mL solution of SA (50mg/mL Gel-SA suspension).

C. Measurement
i) Spot the protein solution (0.5μL) onto a MALDI sample plate.
ii) Layer the Gel-SA suspension (1μL) onto the top of sample spot.
iii) Dry the mixture completely at room temperature under ambient atmosphere (20min~) or in vacuo.

(2) Insoluble Proteins
A. Preparation of a Protein Solution
i) Centrifuge an insoluble protein suspension to obtain a precipitate.
ii) Wash the precipitated protein with Milli-Q water.
iii) Dissolve the protein in 0.5-2% (w/v) of SDS solution and dilute with 10-fold water.
The following procedure is similar as (1)-B and C described above to prepare Gel-SA suspension and the sample is subsequently subjected to MALDI-MS analysis.

(3) Highly Sensitive Detection of Proteins that Contain no SDS (as an application)
Gel-SA-SDS suspension enables extremely sensitive detection of proteins in MALDI-MS

A. Preparation of the Proteins
No special treatment is needed.

B. Preparation of Gel-SA-SDS Suspension
i) Dissolve SA into 50% acetonitrile-0.1% (v/v) TFA to adjust the concentration to 10mg/mL.
ii) Add the ion-exchange silica gel to make a concentration 50mg/mL.
iii) Add 0.5mg (0.05%) of SDS for 1 mL of the suspension (= Gel-SA-SDS suspension)

C. Measurement
i) Spot the protein solution (0.5μL) onto a MALDI sample plate.
ii) Layer the Gel-SA suspension (1μL) onto the top of sample spot.
iii) Dry the mixture completely at room temperature under ambient atmosphere (20min~) or in vacuo.
iv) Acquire the spectrum

Fig. 2 MALDI-TOF Mass Spectra of an Insoluble Protein

■Application
Since this method enables measurement of insoluble proteins without removing SDS, it is expected to apply to membrane protein research.

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S0588 Sodium Dodecyl Sulfate [for Electrophoresis]
D2932 3,5-Dimethoxy-4-hydroxycinnamic Acid [Matrix for MALDI-TOF/MS]