Protein Protocols Protein Protocols

682 Wada
2. Detection of variants through mass measurement of the intact protein molecule with ESIor
MALDI-MS.
3. Chemical or enzymatic cleavage.
4. ldentification of mutated peptides through “peptide mass mapping” of peptide mixtures
using ESI-, MALDI-, or FAB-MS.
5. Characterization of the mutation.
2. Materials
2.1. Sample Preparation
1. LC column (C4, 300 Å; diameter 4.6 mm; length 150 mm) (Vydac, Hestperia, CA), or small
devises packed with RP media such as ZipTip (Millipore, Bedford, MA) or Poros
(PerSeptive Biosystems, Foster City, CA).
2. Trifluoroacetic acid (TFA) (Sigma).
3. Acetonitrile (high-performance liquid chromatography [HPLC]-grade) (Sigma).
2.2. Reduction and Alkylation (Carboxymethylation)
1. Stock solution A: 1 M Tris-HCl containing 4 mM EDTA, adjusted to pH 8.5 with HCl.
2. Stock solution B: 8 M Guanidium chloride.
3. Mix stock solutions A and B at a 1:3 ratio to make a solution of 6 M Guanidium chloride,
0.25 M Tris-HCl, and 1 mM EDTA prior to use.
4. Dithiothreitol (DTT, Sigma).
5. Iodoacetic acid, free acid (Sigma).
2.3. Cleavage
1. Cyanogen bromide (CNBr) (Sigma) (toxic, handle with gloves in hood).
2. Trypsin (N-tosyl-L-phenylalanine chloromethyl ketone [TPCK]-treated) (Sigma type XIII).
3. Lysylendopeptidase (Wako, Osaka, Japan) or endoproteinase Lys-C (Boehringer).
4. Endoproteinase Glu-C (Boehringer).
5. Endoproteinase Asp-N (Boehringer).
6. 50 mM Ammonium hydrogen-carbonate, pH 7.8 (pH adjustment is not required).
7. 50 mM Ammonium carbonate, pH 8.3 (pH adjustment is not required).
8. 50 mM Tris-HCl, pH 7.4.
2.4. Mass Spectrometry
1. Sinapinic acid (10 mg/mL) in acetonitrile–water–TFA (30:69.9:0.1, by vol) for
MALDI matrix.
2. Acetonitrile–water–acetic acid (49:49:2, by vol) or methanol–water–acetic acid
(75:24.8:0.2, by vol) for ESI solvent.
3. Glycerol (Sigma)–trichloroacetic acid (Sigma) (95:5 [w/w]) for FAB matrix.
3. Methods
3.1. Procedures Prior to MS
3.1.1. Purification and Desalting by RP-LC
In general, MS requires no special procedures for sample preparation. However,
removing salts (desalting) is preferred because the formation of adduct ions with alkali
metals such as sodium and potassium will reduce the abundance of the protonated
molecular ion species, which are the informative ions for the mass measurement of