Protein Protocols Protein Protocols

Protein Ladder Sequencing 735
Protein ladder sequencing has the potential to be used in the following biological
applications.
1. In conjunction with peptide fragment mass mapping, the method can provide additional
sequence information and facilitate protein sequence database searching for protein
identification.
2. The method can be used to study the nature and to determine the sites of chemical modifications
and posttranslational modifications (e.g., phosphorylation, and glycosylation).
3. In protein processing and metabolism pathway studies, the method can be used to identify
and confirm the termini of protein processing products.
2. Materials
2.1. Chemicals and Reagents (see Note 1)
1. Phenylisothiocyanate (PITC) (Pierce [Rockford, IL] or Sigma [St. Louis, MO].
2. Phenylisocyanate (PIC), >98% (Aldrich, Milwaukee, WI).
3. Pyridine (Pierce).
4. Hexafluoroisopropanol (HFIP), 99.8+% (Aldrich).
5. Heptane (ABI [Foster City, CA] or Pierce).
6. Ethyl acetate (ABI, Pierce, or Sigma).
7. Trifluoroacetic acid (TFA), anhydrous (Pierce).
8. 12.5% Trimethylamine (TMA) (ABI).
9. α-Cyano-4-hydroxycinnamic acid, 97% (Aldrich).
10. Acetonitrile (ABI).
11. Nitrogen, 99.99% (Matheson, Montgomeryville, PA).
12. Distilled and deionized water (prepared by using Milli-Q UV Plus water purification system).
2.2. Laboratory Equipment
1. Eppendorf Micro-Centrifuge (Brinkmann Instruments, Westbury, NY).
2. Savant SC110 Speed-Vac (Savant Instruments, Farmingdale, NY).
3. Multi-Block Heater (Lab-Line Instruments, Melrose Park, IL).
4. Fisher Vortex (Fisher Scientifics, Pittsburg, PA).
5. In-house laboratory vacuum.
6. Polypropylene microcentrifuge tube (PGC Scientifics, Gaithersburg, MD).
2.3. Mass Spectrometry
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer
(see Note 2).
2.4. Preparing Reagents (see Note 3)
1. Coupling buffer: pyridine/water (1:1 v/v, pH 10.1). 250 µL water, 250 µL pyridine.
2. Coupling reagent: PITC/PIC/pyridine/HFIP (24:1:72:3 v/v). (Prepared under a blanket of
dry nitrogen). 120 µL PITC, 5 µL PIC, 360 µL pyridine, 15 µL HFIP.
3. Cleavage reagent: 500 µL TFA.
4. Conversion buffer: 12.5% TMA/pyridine (1:1 v/v). 100 µL 12.5% TMA, 100 µL pyridine.
5. Conversion reagent: PIC/pyridine/HFIP (1:80:2.5 v/v). 2 µL of PIC, 160 µL pyridine,
5 µL HFIP.
6. Washing solvent A: Heptane/ethyl acetate (10:1 v/v). 10 mL heptane, 1 mL ethyl acetate.
7. Washing solvent B: Heptane/ethyl acetate (2:1 v/v). 8 mL heptane, 4 mL ethyl acetate.