MPC Protocol Overview

Sample Preparation
General Guidelines: Samples submitted for analysis should be processed with extreme care to avoid contamination with human keratin and other possible contaminating proteins. Use appropriate methods for your sample to determine protein concentrations. Total protein content of a cell will vary considerably among cell types. Each sample vial submitted should contain sufficient sample to run the number of gels required. Do not solubilize the samples; all samples are prepared on site. With the exception of gel slices, all samples must be sent frozen or lyophilized.

Sample Types

Physiological fluids
Serum and plasma can be submitted directly and require no further processing. Collect and store at -80oC. Most other body fluids require concentration, desalting or both. Centricon units (Millipore) or equivalent devices can be used.

Chromatography Fractions or
Partially Purified Protein Preparations
Reduce the salt concentration by appropriate methods. The total amount of salt should not exceed 20 mmol for a gel sample. The maximum volume cannot exceed 100 ml.

Whole Cell Lysates and Membrane Proteins
Whole cells submitted for analysis should be pelleted by centrifugation and washed to remove growth media. Freeze cells immediately and store at -80oC. Protein estimations are based on the following generalizations. Whole cell pellets are estimated to
be ca. 7% protein. Membrane proteins are estimated at 10% of the total protein or 0.7% of the whole cell pellet. Weight of whole cell pellet is determined from wet weight of cells assuming a density of 1g/ml. Alternate calculation for 1 ml packed cell volume: prokaryotes: 0.5-2 x 107 cells; eukaryotes: 0.5-2 x 106 cells. Cells grown in monolayer can be harvested by scraping. Trypsinize a representative culture for cell counting or protein determination.

Subcellular Fractions
Pellet nuclei, mitochondria, microsomes, etc. as above for whole cells. Cytosolic protein preparations should contain no
more than 50 mM salt.

Gel Slices
Gel slices submitted for analysis should not exceed 1 mm in
width. Typically the smaller the plug the better for peptide extraction.
Store gels plugs in HPLC grade water at 4oC. Note: Not all staining
methods are fully compatible with mass spectroscopy analysis. Protocols for compatible Colloidal Coomassie (250 ng/mm2 or 30 ng/band), Silver (5-10 ng/mm2 or 1.5 ng/band) and Fluorescence stains are available.

2-D Gel Analysis

Standard format for second dimension analysis is a 20 cm x 20 cm and 1.5 mm thick SDS-PAGE gel, requiring an 18 cm Immobolized pH
Gradient (IPG) strip (Amersham Pharmacia Biotech) for the first
dimension. The initial staining method is dependent on the amount of
sample provided. Reimaging with increasing sensitivity of staining is
available.

Typically 1-3 mgs of a complex protein mixture is needed for each
preparative 2-D gel for staining by Colloidal Coomassie Stain a variation of the Bradford Method (Bradford, 1976). Coomassie stain sensitivity is generally 250 ng/mm2 or 30 ng/band. Increased sensitivity can be achieved with Silver Stain (adapted from Mortz, et al., 2001). Silver staining requires a minimum of 300 mg of initial sample for each gel and typically each protein is visualized at 5-10 ng/mm2 or 1.5 ng/band. Fluorescence staining is the most sensitive stain and is available for low-level detection. Each gel requires 100-300mg of initial sample. Gels are stained with ruthenium II tris
(bathophenanthroline disulfonate (RuBPS) as adapted from Rabilloud et al., 2001.

Following staining all gels are then scanned using either a flatbed scanner in transmissive mode or using a Molecular Imager FX Pro Plus (BioRad) scanner for fluorescence gels. The gels are then wrapped in Saran wrap and stored at 4oC. Fluorescent stained gels are placed in amber Zip-lock bags to prevent photobleaching.

In-Gel Digestion

Automated spot picking is performed by a robotic system (Ettan Spot
Picker, Amersham Pharmacia Biotech). Manual excision of gel spots (2D) and bands (1D) are performed with a dermal punch or a razor blade. All samples are placed in 96 well microtiter plates with HPLC grade water and stored at 4oC. Tryptic digestion is performed using a MassPrep Robotic Workstation (MicroMass, UK). A robotic or manual in-gel digestion protocol is available. Tryptic peptides are typically extracted with 1% formic acid, 2% acetonitrile in water followed by a second extraction with 50% acetonitrile. In some instances, ZipTip (Millipore) purification and concentration of the extracted tryptic digest is performed as per the manufacturer’s directions. The MassPrep robot can also be used for the Zip tipping and can also spot samples on the MALDI target plate for mass spectrometric analysis.

Mass Spectrometry Analysis

Tryptic digests are analyzed using matrix-assisted laser
desorption/ionization (MALDI) mass spectrometry. The matrix is a-cyano, 4-hydroxy cinnamic acid. A positive ion mass spectrum is obtained for the tryptic digest using one of three mass spectrometers: MALDI-QTOF (MicroMass, UK), DE Pro (Applied Biosystems), or a TOF-TOF (4700 Proteomics Analyzer, Applied Biosystems). The DE Pro and TOF-TOF are operated in reflectron mode. The MALDI Q-TOF and DE Pro mass spectrometers use a N2 laser at 354 nm and 20 Hz. The TOF-TOF is a Nb:YAG laser operated at the third harmonic (355 nm) and 200 Hz for high through-put operations. Spectra are calibrated using internal calibration using autodigestion products of trypsin when possible; otherwise
external calibration is used. Mass accuracy is better than 50 ppm, usually 10 ppm. Instrument sensitivity is of the order of 10 fmol or better with all three instruments. Peptide masses are submitted to SwissProt or NCBInr databases for peptide mass fingerprint (PMF) protein identification of the protein, using Protein Prospector.

In instances where a positive protein identification cannot be made using PMF, selected peptide ions in the digest are analyzed by ms/ms analysis using either the MALDI-QTOF or the TOF-TOF. In general, three peptide ions are selected for MS/MS analysis. Fragment ion masses are submitted to the database for identification using MASCOT or Protein Prospector. Automated database searching using mass spectrometry data from the TOF-TOF (MS or MS/MS data) is done using GPS software using the MASCOT search engine (AppliedBiosystems).

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