PhyNexus has developed an automated process to desalt or “clean-up” proteins/peptides using C18 columns and C4 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. With the high capacity and versatility in protein binding, the PhyNexus C18 column is the optimal choice in developing a fluid MALDI or LC-MS workflow.
|C18 & C4 tips are available in tip volumes (µL) of 200|300|500|1000 and resin volumes (µL) of 5|10|20|40|80|160|320 depending upon robot configuration.|
C18 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. When comparing the capacity of some of the leading C18 tips, the C18 PhyTip column binds more than double the amount of protein per mL of resin while exhibiting a comparable recovery and range.
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.
C18 tips with our proprietary Dual Flow Chromatography provide complete antibody binding and high final concentrations. C18 PhyTip columns are compatible with most 8, 12, and 96-channel automation liquid handling robots including Agilent Technologies, Beckman Coulter, Dynamic Devices, Hamilton, Opentrons, Perkin Elmer, and Tecan.
PhyNexus has developed an automated process to desalt or “clean-up” proteins/peptides using C18 & 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 C18 resins to capture and separate peptides based on their hydrophobicity.
The PhyTip column is composed of C18 & 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.
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.
New Advances towards Quantitative Proteomics: Optimization of Peptide Labeling by Fluorescent Isotope Coded Affinity Tag (FCAT)
Z. Rivera, M. Olajos, T. Ringer, A.R. Mayer, G. K. Bonn and A. Guttman (Horvath Laboratory of Bioseparation Sciences, Austria)
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