Poly-L-ornithine cleanup for MALDI analysis
Poly-L-ornithine is widely used to coat plasticware and glass surfaces to enhance cell attachment. PEGylated poly-L-ornithine is also used in drug delivery. The method below utilizes ZipTip® C4 micropipette tips as part of poly-L-ornithine stability testing; they were used for salt removal prior to MALDI-TOF MS analysis.
Poly-L-Ornithine Reconstitution and Incubation
- Dissolve poly-L-ornithine in 0.9% saline at a concentration of 1 mg/mL (stirring at 300 rpm for 15 min).
- Create seven 100 µL aliquots and store at -20°C until incubation.
- Incubation is performed as follows:
- Seventy-two hours prior to analysis, thaw two aliquots. Place one aliquot in an incubator set to 25°C, and a second aliquot in an incubator set to 40°C.
- Twenty-four hours prior to analysis, thaw two aliquots. Place one aliquot in an incubator set to 25°C, and a second aliquot in an incubator set to 40°C.
- Eight hours prior to analysis, thaw two aliquots. Place one aliquot in an incubator set to 25°C, and a second aliquot in an incubator set to 40°C.
- At the time of analysis, thaw one aliquot to use as a control sample.
ZipTip® Cleanup
- Place 10 µL of each sample in a separate microcentrifuge tube.
- Wet the ZipTip® (C4) micropipette tip by 3x aspiration of acetonitrile, with disposal to waste.
- Equilibrate the ZipTip® micropipette tip by 3x aspiration of distilled water, with disposal to waste.
- Aspirate 10 µL sample volume into the ZipTip® micropipette tip and cycle ten times, with final disposal to waste.
- Wash the ZipTip® micropipette tip by 5x aspiration of distilled water, with disposal to waste.
- Place 10 µL of 50% acetonitrile/50% distilled water in clean microcentrifuge tubes (one tube for each sample). Use the solution to aspirate and dispense eluant through the ZipTip® micropipette tip; cycle ten times.
MALDI-TOF Analysis
A. Solution Preparation – System Suitability Standards
- Prepare a volume of α-cyano-4-hydroxycinnamic acid at a concentration of 10 mg/mL in 50% acetonitrile/0.05% Trifluoroacetic Acid in distilled water.
- Prepare insulin oxidized β-chain, insulin, cytochrome C, and apomyoglobin at a concentration of 10 pmol/µL in an appropriate solution.
- Dissolve insulin oxidized β-Chain in 50% acetonitrile/0.05% trifluoroacetic acid in distilled water.
- Dissolve insulin in 1.0% trifluoroacetic acid in distilled water.
- Dissolve all other standards in 0.1% trifluoroacetic acid in distilled water.
- Mix calibrant solutions at a 1:1 (v:v) ratio with the prepared 10 mg/mL α-cyano-4-hydroxycinnamic acid solution, spotted on the MALDI target, and allow to dry. Mix matrix and samples in a 1:1 ratio.
- For 5,000 – 11,000 Da molecular weight poly-L-ornithine samples, use a 1:3:9 (v:v) mixture of insulin oxidized β-chain:insulin:cytochrome C as a calibrant.
- For 11,000 – 17,000 Da molecular weight poly-L-ornithine samples, use a 1:3:9 (v:v) mixture of insulin:cytochrome:apomyoglobin as a calibrant.
- Utilized ratios of calibrants may change as needed to provide for increased ionization of the larger molecular weight standards.
B. Solution Preparation – Poly L-Ornithine
- Prepare a second volume of α-cyano-4-hydroxycinnamic acid at a concentration of 3.8 mg/mL in methanol.
- Mix desalted samples at a 1:1 volume ratio with the prepared 3.8 mg/mL α-cyano-4-hydroxycinnamic acid and spot on the MALDI target in triplicate.
- 3Dry samples on target and place into the MALDI-TOF instrument for analysis.
C. System Suitability
- Calibrate the instrument using the appropriate calibrant mixture.
- Verify system suitability by analyzing the cytochrome C molecular weight error.
D. Sample Analysis
- Use MALDI-TOF instrumental settings typically used for analysis of poly-L-lysine to test for suitability with the poly-L-ornithine (poly-L-lysine is similar in size and composition to poly-L-ornithine).
- Adjust instrumental parameters as required to obtain quality spectra.
- After instrumental parameters are optimized, analyze three sample spots of each time point.