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

ZipTip^® Micropipette Tips

Bed Volume	Sorbent
	C18	C4	SCX
0.6 µL	ZTC18S	ZTC04S	ZTSCXS
0.2 µL	ZTC18M	----	----

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

1. Dilute sample to a final volume of 10 µL in 0.1 M TEAA.
2. Attach ZipTip^® (C18) tip to appropriate pipette, set to a maximum of 10 µL.
3. 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.
4. Equilibrate the ZipTip^® tip by washing 3x with 10 µL of 0.1 M TEAA.
5. 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.
6. Wash ZipTip^® tip 3x with 10 µL of fresh 0.1 M TEAA, dispensing to waste after each aspirate-and-dispense cycle.
7. Wash ZipTip^® tip 3x with 10 µL of dH₂O, dispensing to waste after each aspirate-and-dispense cycle.
8. 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. 

1. Aspirate and dispense eluant through ZipTip^® pipette tip at least 3 times without introducing air.

Sample/MALDI Target Preparation

1. Mix matrix and samples in a 1:1 ratio.
2. 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

1. Dissolve poly-L-ornithine in 0.9% saline at a concentration of 1 mg/mL (stirring at 300 rpm for 15 min).
2. Create seven 100 µL aliquots and store at -20°C until incubation.
3. Incubation is performed as follows:
   1. 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.
   2. 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.
   3. 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.
   4. At the time of analysis, thaw one aliquot to use as a control sample.

ZipTip^® Cleanup

1. Place 10 µL of each sample in a separate microcentrifuge tube.
2. Wet the ZipTip^® (C4) micropipette tip by 3x aspiration of acetonitrile, with disposal to waste.
3. Equilibrate the ZipTip^® micropipette tip by 3x aspiration of distilled water, with disposal to waste.
4. Aspirate 10 µL sample volume into the ZipTip^® micropipette tip and cycle ten times, with final disposal to waste.
5. Wash the ZipTip^® micropipette tip by 5x aspiration of distilled water, with disposal to waste.
6. 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

1. 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.
2. Prepare insulin oxidized β-chain, insulin, cytochrome C, and apomyoglobin at a concentration of 10 pmol/µL in an appropriate solution.
   1. Dissolve insulin oxidized β-Chain in 50% acetonitrile/0.05% trifluoroacetic acid in distilled water.
   2. Dissolve insulin in 1.0% trifluoroacetic acid in distilled water.
   3. Dissolve all other standards in 0.1% trifluoroacetic acid in distilled water.
3. 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.
   1. 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.
   2. 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.
   3. 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

1. Prepare a second volume of α-cyano-4-hydroxycinnamic acid at a concentration of 3.8 mg/mL in methanol.
2. 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.
3. 3Dry samples on target and place into the MALDI-TOF instrument for analysis.

C.        System Suitability

1. Calibrate the instrument using the appropriate calibrant mixture.
2. Verify system suitability by analyzing the cytochrome C molecular weight error.

D.      Sample Analysis

1. 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).
2. Adjust instrumental parameters as required to obtain quality spectra.
3. 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.

1. Place 10 µL of each sample in a separate microcentrifuge tube.
2. Wet the ZipTip^® (C4) micropipette tip by 3x aspiration of acetonitrile, with disposal to waste.
3. Equilibrate the ZipTip^® micropipette tip by 3x aspiration of distilled water, with disposal to waste.
4. Aspirate 10 µL sample volume into the ZipTip^® micropipette tip and cycle ten times, with final disposal to waste.
5. Wash the ZipTip^® micropipette tip by 5x aspiration of distilled water, with disposal to waste.
6. 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) https://doi.org/10.1016/j.mcpro.2023.100532	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) https://doi.org/10.1002/jssc.202300064	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