Cleanup and Concentration of Trace-level Analytes from Complex Matrices Using ZipTip® Micro-SPE Pipette Tips
- Sample preparation with ZipTip® micropipette tips
- Oligonucleotide cleanup for MALDI analysis
- Poly-L-ornithine cleanup for MALDI analysis
- General cleanup protocol for mAbs, non-toxic ADCs, and other mAb conjugates for MALDI analysis
- Select publications for ZipTip® micropipette tips
Sample Preparation with ZipTip® Micropipette Tips
- Samples for mass spectrometry (MS) analysis must be adequately cleaned up and concentrated to get optimal results.
- MALDI-TOF MS is used to analyze intact large molecules such as proteins, monoclonal antibodies, peptides, oligonucleotides, and polymers.
- Nano LC-MS offers excellent sensitivity for small volumes of diluted samples. It is used in omics research and other applications where sample such as cerebrospinal fluid (CSF) is limited.
- ZipTip® micropipette tips are 10 μL pipette tips with a microvolume bed of chromatography medium fixed at the end. They are designed for purifying and concentrating femtomole to picomole amounts of proteins and peptides, oligonucleotides, and polymers, providing improved mass spec data.
- ZipTip® micropipette tips are available in C18, C4, and strong cation (SCX) resins, with either a 0.6 or 0.2 µL bed.
- C18 is widely used in peptide analysis/protein digest
- C4 is recommended for large molecules
- SCX works especially well for tryptic peptides that have a minimum of two positive charges per molecule at acidic pH
Oligonucleotide Cleanup for MALDI Analysis
This protocol uses ZipTip® micropipette tips with C18 resin to clean up oligonucleotide samples prior to MALDI. Use 100 – 200 ng oligonucleotide sample for cleanup.
ZipTip® Cleanup
- Dilute sample to a final volume of 10 µL in 0.1 M TEAA.
- Attach ZipTip® (C18) tip to appropriate pipette, set to a maximum of 10 µL.
- Prewet the ZipTip® micropipette tip by depressing the pipettor plunger to a dead stop. Aspirate 50% acetonitrile (ACN) in dH2O into the tip, dispense to waste, and repeat.
- Equilibrate the ZipTip® tip by washing 3x with 10 µL of 0.1 M TEAA.
- Bind sample to the ZipTip® tip by pressing the pipettor plunger to a dead stop. Place tip in the sample and perform an aspirate-and-dispense cycle 5 – 10 times.
- Wash ZipTip® tip 3x with 10 µL of fresh 0.1 M TEAA, dispensing to waste after each aspirate-and-dispense cycle.
- Wash ZipTip® tip 3x with 10 µL of dH2O, dispensing to waste after each aspirate-and-dispense cycle.
- Dispense anywhere from 1-10 µL of 50% ACN in dH2O into a clean microcentrifuge tube using a standard pipette tip. The larger the volume used, the better the recovery (at the expense of concentration).
NOTE: If sample concentration is a concern, dispense the sample into prepared matrix instead of the 50% ACN. This avoids the further dilution that occurs in mixing with matrix after elution.
- Aspirate and dispense eluant through ZipTip® pipette tip at least 3 times without introducing air.
Sample/MALDI Target Preparation
- Mix matrix and samples in a 1:1 ratio.
- Spot 1 µL sample 3x on the MALDI target and allow spots to dry.
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.
General cleanup protocol for mAbs, non-toxic ADCs, and other mAb conjugates for MALDI analysis
ZipTip® with C4 resin is recommended for proteins with a molecular weight >100 kDa, which makes them ideal for cleaning up monoclonal antibodies.
- 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% ACN with 0.05% TFA in 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.
NOTES:
- If sample concentration is a concern, dispense the sample into prepared matrix (instead of the 50% ACN with 0.05% TFA in water) as these molecules need a higher concentration to properly ionize due to their large size.
- Sinapic acid is used for the matrix.
Select Publications for ZipTip® Micropipette Tips | ||||
---|---|---|---|---|
Year | Title (Journal) | Sample | ZipTip® Resin | Downstream Analysis |
2023 | Phenotypic Heterogeneity Analysis of APC-Mutant Colon Cancer by Proteomics and Phosphoproteomics Identifies RAI14 as a Key Prognostic Determinant in East Asians and Westerners (Molecular and Cellular Proteomics) | Colon cancer | C18 | Nano LC-MS |
2023 | Proteotypic peptides of hairs for the identification of common European domestic and wild animal species revealed by in-sample protein digestion and mass spectrometry analysis (J of Separation Science) | Animal hair | C18 | MALDI-TOF MS |
2023 | Glycomics-Assisted Glycoproteomics Enables Deep and Unbiased N-Glycoproteome Profiling of Complex Biological Specimens https://link.springer.com/protocol/10.1007/978-1-0716-2978-9_16 | Serum Plasma | C18 | Nano LC-MS |
2023 | Quantification of Serum Metabolites in Early Colorectal Adenomas Using Isobaric Labeling Mass Spectrometry https://doi.org/10.1021/acs.jproteome.3c00006 | Serum | SCX | Nano LC-MS |
2023 | Changes in Serum Protein–Peptide Patterns in Atopic Children Allergic to Plant Storage Proteins https://doi.org/10.3390/ijms24021804 | Serum | C18 | MALDI-TOF MS |
2023 | A proteomic profile of the healthy human placenta https://doi.org/10.1186/s12014-022-09388-4 | Placenta | C18 | Nano LC-MS |
2023 | Purification and activity evaluation of novel anti-inflammatory peptides from pearl oyster (Pinctada martensii) hydrolysates https://doi.org/10.1039/D2FO04046H | Pearl oysters | C18 | Nano LC-MS |
2022 | Maternal serum proteomic profiles of pregnant women with type 1 diabetes https://doi.org/10.1038/s41598-022-12221-5 | Serum | C18 | MALDI-TOF MS Nano LC-MS |
2022 | A subtractive proteomics approach for the identification of immunodominant Acinetobacter baumannii vaccine candidate proteins https://doi.org/10.3389/fimmu.2022.1001633 | Bacterial culture | C18 | Nano LC-MS |
2022 | Using Activity-Based Proteomics for the Quantification of Deubiquitinases in Animal Tissue https://doi.org/10.1007/978-1-0716-2803-4_4 | Animal tissue | C18 | Mass Spec |
2022 | Phosphotyrosine Profiling Using SILAC https://doi.org/10.1007/978-1-0716-2863-8_9 | Cell culture | C18 | Mass Spec |
2022 | Composition and abundance of midgut surface proteins in the Asian citrus psyllid, Diaphorina citri https://doi.org/10.1016/j.jprot.2022.104580 | Insect | C18 | timsTOF |
2022 | Whole Shotgun Proteomics and Its Role in Mycoremediation https://doi.org/10.1007/978-1-0716-2006-9_16 | Fungus (Mycellium) | C18 | Nano LC-MS |
2022 | The Antibody Dependant Neurite Outgrowth Modulation Response Involvement in Spinal Cord Injury https://doi.org/10.3389/fimmu.2022.882830 | Spinal cord | C18 | Nano LC-MS |
2021 | Proteomic Profiling of Cerebrospinal Fluid by 16-Plex TMT-Based Mass Spectrometry https://doi.org/10.1007/978-1-0716-1936-0_3 | CSF | C18 | Nano LC-MS |
2021 | Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry https://doi.org/10.1007/978-1-0716-1190-6_3 | RAS protein isoforms | C4 | Nano LC-MS |
2021 | A proteomics approach to characterizing limited hydrolysis of whey protein concentrate https://doi.org/10.1016/j.foodchem.2021.129235 | Whey protein concentrate | C18 | MALDI-TOF MS |
2021 | Cell Wall Proteome Profiling of a Candida albicans Fluconazole-Resistant Strain from a Lebanese Hospital Patient Using Tandem Mass Spectrometry—A Pilot Study https://doi.org/10.3390/microorganisms9061161 | Fungal culture | C18 | MALDI-TOF MS |
2021 | Mass spectrometry-based top-down and bottom-up approaches for proteomic analysis of the Moroccan Buthus occitanus scorpion venom https://doi.org/10.1002/2211-5463.13143 | Scorpion venom | C4 | Nano LC-MS |
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