Protocols

Band or Spot Excision

Spots may be picked from 2D-gels and deposited into the wells of a microtiter plate using the in-house robotic spot picker located in Farber 123. Alternatively, spots may be picked manually as outlined below. Note that the following section describes excision of bands from a 1D-gel; for spot picking from a 2D-gel, a single plug from the center of the spot is the best approach.

• Prewash microfuge tubes three times using a solution containing 0.1% TFA/60% acetonitrile.

• Excise three plugs per band using a syringe with blunt needle (1.7-2.0 mm ID) or blunted pipettor tip. Control plugs can be excised from an unstained region of the gel.

• Estimate gel volume of each plug by eye or by weighing.

Reduction/Alkylation

Reduction and alkylation blocks free sulfhydryl groups and can greatly clarify ensuing MALDI spectra. (Note that alkylation adds 58Da per SH). Reduction and alkylation is typically helpful, but not always essential. Samples derived from 2D-DIGE applications are reduced and alkylated between the IEF and SDS steps, and thus this procedure is unnecessary.

Note: all solutions containing acetonitrile (AcCN) with NH4HCO3 should be freshly prepared.

• To destain, wash gel plug for 30-60 min at 37° in 100-200µL 200mM NH4HCO3 containing 50% AcCN
• Remove the solution. If necessary, repeat the above step to remove all stain.
• Add 25 uL of 10 mM DTT/25 mM NH4HCO3 solution to the destained gel plug, incubate for 15 minutes at 65°C.
• Return tubes to RT! (5 minutes), add iodoacetamide until the solution is 10 mM.
• Incubate for 30 minutes in the dark.
• Remove the solution.
• Wash the gel plugs with 250 uL of 25 mM NH4HCO3, incubate for 15 minutes at RT.
• Vortex gently, Remove the solution.
• Wash the gel plugs with 250 uL of water, incubate for 5 minutes at RT.
• Vortex gently, Remove the solution.

In-Gel Digestion and Peptide Isolation

Note: all solutions containing acetonitrile (AcCN) with NH4HCO3 should be freshly prepared.

• Estimate gel volume, either by eye or by weighing
• To destain, wash gel plug for 30-60 min at 37° in 100-200µL 200mM NH4HCO3 containing 50% AcCN (This step can be eliminated if above reduction/alkylation protocol was used).
• Remove wash solution and dry gel plug on speed-vac for ~1h
• Add approximately one gel volume of 20µG/mL trypsin in 40mM NH4HCO3 containing 10% AcCN. Incubate 60 min at RT
• Add 30-50µL 40mM NH4HCO3 containing 10% AcCN and incubate O/N at 37°
• Vortex sample.

Sample preparation for MALDI analysis

Matrix: The two most commonly used matrices are alpha-cyano-4-hydroxycinnamic acid (alpha-CN) and sinapinic acid (SA). Sinapinic acid is a "hotter" matrix recommended for samples in the mass range >3.5kD; for most applications, alpha-CN is preferred because it yields cleaner backgrounds. alpha-CN is dissolved at 30 mG/mL in 0.1% TFA/60% AcCN. SA can be prepared at 50mG/mL in 0.1% TFA/60% AcCN.

• Wash the MALDI plate with MeOH, followed by Milli-Q water, and AcCN, successively.
• The supernatant may be concentrated by speed-vac to dryness, and then brought up in no more than 5-10µL 0.1% TFA/60% AcCN. Digests derived from low abundance samples may be dissolved in 1µL. 0.5 µL of this peptide solution should be spotted over 0.5 µL of pre-spotted matrix for MALDI analysis.
• Alternatively, sample clean up can be achieved using ZipTipµC18, from Millipore Instructions for ZipTipµC18 use are as follows:

• Wet a ZipTip (Millipore) by drawing up and expelling 2x10µL 100% AcCN.
• Equilibrate ZipTip by drawing up and expelling 3x10µL 0.1% TFA.
• Pull sample into ZipTip and expel back into tube; repeat 10x.
• Wash by drawing up and expelling 3x10µL 0.1% TFA.
• Elute sample into a clean microfuge tube with 5µL 0.1% TFA/60% AcCN.
• Spot 0.5 µL onto the MALDI target, pre-spotted with 0.5 µL matrix.
• Alternatively, for a more concentrated sample, elute from ZipTip directly onto target with 1 µL matrix solution.

MALDI-TOF Analysis

It is essential that all spots on the target be completely dry before the target is inserted into the sample chamber. Moisture on the target leads to rapid degradation of the electrodes!

The key to a successful MALDI-TOF analysis lies in accurate calibration. This can be accomplished either externally or internally. For external calibration, we typically use a standard peptide mix that can be purchased from Bruker (see www.bdal.de), ideally spotted very near to the samples being analyzed. Alternatively, internal calibration may be used. This relies on the endogenous presence of two autolysis products of porcine trypsin, having MWs of 842.510 and 2211.105, in each sample. Because both these products are not invariably present, it is safest to include external calibrants in your experiment.

Calibration is best done using the lowest possible laser energy with the instrument in reflector mode. Under these conditions, all of the peptides mentioned above should yield a cluster of 3 peaks resolved to or near baseline, and separated by 1 amu. This is referred to as a monoisotopic spectrum. The calibrant peak is the lowest of each group.

Maldi is a very sensitive technique, allowing detection at the low fmole level. At the same time, it is important to remember that Maldi data are often quite concentration-dependent. Spotting too much sample can cause degradation of the signal. Peak broadening is often one indication of this. If this problem is suspected, it is useful to spot the sample both neat, and at 1:10 dilution, and compare the resulting spectra. "Ideal" sample loads are ~ pM, with sensitivities to the fM range.

Database Searches

Maldi spectra are compared to available databases on-line using the BioTools module of the Maldi operating software. The default submission site is Matrix Science's MASCOT. Upon entering the MASCOT site, the user is prompted to provide information about their sample (ie, approx MW, state of cysteine residues, post-translational modifications to be considered, enzyme used for digestion) and the parameters of their search (which database or subset thereof, number of allowed missed cleavages, data stringency). The form can be found at:

http://www.matrixscience.com/cgi/index.pl?page=../home.html

The first set of parameters is obviously user-dependent. For the others, we routinely use the NCBI non-redundant database (limited by species as desired), and a maximum of two missed cleavages. Stringency is empirical: the higher the better. In general, start at 100ppm and examine results, then move higher or lower as desired.

Materials

Sequencing Grade Modified Porcine Trypsin, Promega catalog #V5111

A vial containing 20µG can be resuspended in 1 mL 40mM AmHCO3 containing 10% AcCN and stored at -20° for several months. Trypsin can also be stored frozen in 1mM HCl, under which conditions it is stable almost indefinitely. If you choose to store your trypsin in HCl, it is important to ensure that your digestion buffer is capable of neutralizing the acid.

ZipTipµC18, Millipore, catalog #ZTC18M096

Matrices:

a-Cyano-4-hydroxycinnamic acid (a-CN), Bruker Daltonics, catalog #554389/203072. Prepare a saturated solution (>10mG/mL) in 60% AcCN, 0.1% TFA. Can be stored several days in refrigerator.

Sinapinic acid (3,5-dimethoxy, 4-hydroxycinnamic acid), Sigma catalog #D7927. Prepare a saturated solution in 60% AcCN. Can be stored in refrigerator for at least 30 days.