PhyNexus has developed an automated process to desalt or “clean-up” proteins/peptides using C4 PhyTip columns in order to streamline sample preparation for mass spectrometry analysis (up to 96 at a time). When preforming a reversed phase purification using a manual pipette, back pressure in aspirating solutions can cause an inconvenience to the operator and inconsistencies within results. PhyTip columns perform the clean-up with the consistency, ease, and scalability that go hand-in-hand with automation.

PhyTip columns have increased selectivity for polar and non-polar molecules compared to other columns due to the increased bed size and capacity of the PhyTip pipette tip column. This larger bed size will push the equilibrium of the capture to increased amount of capture product.

C18 columns are more hydrophobic than C4 columns providing outstanding sensitivity and high efficiency binding of peptides. Both medium 90 Å and large 300 Å pores are available for differential selectivity of peptides and protein fragments. When choosing between C4 and C18 resin, the major difference is that C4 is slightly more polar than C18 due to its shorter length and closer proximity to the polar silica substrate. Therefore, the selectivity of the C4 resin is increased for more polar molecules.

The columns bind a wide range of protein and polypeptide sizes. The absolute binding capacity is high with a low loss of smaller or more polar polypeptides. The sample is recovered in common mass spectrometry compatible solvents. In addition, gel filtration columns may be used to perform rapid and completely automated 96 at-a-time buffer desalting or buffer exchange.

C4 columns with our proprietary Dual Flow Chromatography provide complete antibody binding and high final concentrations. PhyTip columns are compatible with most 8, 12, and 96-channel automation liquid handling robots including Agilent TechnologiesBeckman CoulterDynamic DevicesPerkin ElmerHamilton, and Tecan.

Description

PhyNexus has developed an automated process to desalt or “clean-up” proteins/peptides using C4 PhyTip® columns for use in mass spectrometry (MS) analysis. PhyTip columns bind proteins/peptides through reversed-phase solid phase extraction (SPE), which utilizes long carbon chain C4 resins to capture and separate peptides based on their hydrophobicity.

The PhyTip column is composed of C4 resins retained on a pipette tip by two thin, inert frit screens. Depending on the downstream assay, the PhyTip column can be made with either 90 Å or 300 Å pore resin in a 5 µL bed and has capacity to process up to 200 µL of solution. The PhyTip column processes peptides through dual flow chromatography, a systematic process of back and forth sample flow performed by a liquid handler that controls the volume and flow rates.

As a result, the method can be utilized to clean-up small to mid-scale peptide samples (up to 96 at a time) for use in matrix-assisted laser desorption-ionization (MALDI) or electrospray ionization (ESI) mass spectrometry as part of an automated process.

Application
Sample Processing on the PhyNexus MEA Personal Purification System
1. Sample prepared to a 0.1 % Trifluoroacetic Acid (TFA) concentration
2. Wetting with 100 µL 100% Acetonitrile (ACN) for 4 cycles
3. Equilibration with 100 µL 0.1 % TFA for 1 cycle
4. Capture with 185 µL sample for 4 cycles
5. Wash with 100 µL of 0.1 % TFA for 1 cycle
6. Elution with 100 µL of 70% ACN + 0.1 % TFA for 4 cycles
Each cycle consists of one aspiration and one expulsion at a flow rate of 1 mL/min. Each elution aspirate/expel step is performed in 4 increments with 5 second pauses, while all other aspirate/expel steps are performed in 8 increments with 30 second pauses. The processing was designed to imitate manual pipetting while compensating for backpressure. The elution plate is sealed to prevent the volatile elution buffer from evaporating during the processing period, and solutions were vacuum degassed to further ensure air does not enter the column.
Documents
Compatibility

Mass Spectrometry PhyTip Columns are available for these robotic systems.

Citations

Posters


A flexible platform for automated high-performance protein purification using micro scale separation technology
J. Lambert, M. Anderson, D. Gjerde, L. Nguyen (PhyNexus)

Innovative Solutions for Automated Protein and Nucleic Acid Purification
Lee Hoang, PhyNexus, Inc., PhyNexus Webinar

Automated, High Throughput Protein Purification and Sample Prep Using PhyTip Columns for Therapeutic Leads Screening and Process Development
Lee Hoang, PhD., PhyNexus, Inc., The 5th Annual Proteins Congress 2012, London

Optimization of Protein Purification Using Small-Scale Separation Columns
Chris Suh, PhD., PhyNexus, Inc., Protein Therapeutics Discovery and Development

PhyNexus Users Group Symposium 2014
Douglas Gjerde, PhyNexus
Lee Hoang, PhyNexus

Select your resin, robotic system, tip and resin volume and see the results in the table below.