Current proteomic protocols require the separation of proteins by either chromatographic or electrophoretic means prior to analysis by mass spectroscopy. Gel electrophoresis protocols suffer from the process of gel extraction and manual processing prior to spotting on MALDI plates for analysis. Reverse phase HPLC is effective at separating protein based on hydrophobic interaction (HIC) and is automation friendly, however, it suffers from the fact that the isolated fractions have a variable concentration of the organic solvents. The buffer/organic solvent mixtures result in incomplete and variable peptide digestion following chromatographic separation.
The proposed method circumvents these difficulties by allowing plasma to be separated using HIC chromatography followed by protein capture and recovery with ion exchange membranes. The recovered proteins are then concentrated using ultrafiltration if needed resulting in ninety-six reproducible fractions fully capable of enzyme digestion and subsequent analysis. The use of a fraction collector and multi-well plate format allows automated protocols to be developed.
Research in proteomics requires a variety of analytical tools for the advancement of knowledge about the proteins involved in biological processes. Current protocols rely heavily on aging technologies such as two-dimensional gel electrophoresis.These technologies allow the protein population in an organism to be separated into classes based on a combination of the pI (isoelectric point) and mass of a specific molecule. This process is labor intensive, difficult to automate and often makes if difficult to interpret data due to the interference of the more abundant but less interesting proteins.
A second less widespread tool for the separation of proteins prior to analysis relies on chromatographic separation using HPLC chromatography. Protein pools and whole plasma are digested, run on HPLC and analyzed by MALDI (mass spectroscopy) after spotting.
This process is more capable of automation but results in a very complex analysis pattern where digested fragments for a single protein may reside in more than one HPLC fraction.
Alternatively, the intact proteins themselves can be separated and spotted directly but the inability to digest the proteins following HPLC complicates the analysis of the subsequent
MALDI analysis. Simplification of this process could be achieved by separating the proteins via chromatography followed by trypsin digestion and analysis on MALDI.
To address the needs of the researchers who are interested in taking a more automated approach to protein separation, HPLC can be used to fractionate the serum proteins along the first dimension based on hydrophobic interaction chromatography followed by a transfer to a second analytical process such as mass spectroscopy. The research gap filled by ion exchange results from the organic solvents in the protein fractions collected from the HPLC run. Organic solvents are known to interfere with further processing such as trypsin digestion. Collection and concentration using a combination of ion exchange and ultrafiltration allows the use of enzyme digestion on the protein fraction after the complexity has been limited to a subset of proteins. Further, the use of multi-well filtration plates containing Pall’s Mustang ™ ion exchange membranes allows the development of automated protocols for the recovery of proteins from organics solvents.
Figure 1
Development of an HPLC Protocol
Calf serum was used to develop a separation protocol that would be used in subsequent protocol development. Fraction collection (see photo) allowed the capture of 1 ml fraction at 1 min each from 20 to 116 min. Note, the act of fractionation acts as an effective albumin depletion for samples prior to 40 min and after 60 min.
Multi-well Fraction Collector
BSA alone
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Figure 2
Optimization of BSA Recovery on Mustang ™ Q Membrane
Based on the highly negative charge at neutral pH for BSA, it is expected that BSA will bind best to Mustang Q chemistry. Dilution of ACN and buffer was needed since the HPLC solution is slightly acidic. At a lower HEPES concentration (5 mM) the BSA ends up in the flow through indicating its inability to bind in ACN at lower buffer concentrations (negative values on the plot represent the concentrations recovered in the flow through) .
At 10 mM HEPES (pH 7.0) the binding and recovery of BSA is near 100% in both 10% and 25% ACN. The positive control on the far left is BSA loaded in 5 mM HEPES without
ACN in the solution. The data show the mean of n=8 samples.
BSA Recovery with
Mustang Q Membrane
HE PES 5 HE PES 10
0.2 m g /m l BSA
10% AC N - 1
10% AC N - 2
10% AC N FT
25% AC N - 1
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25% AC N FT
Figure 3
BSA Recovery is Low Using Mustang S Membrane
It is not expected that BSA will bind to the negatively charged Mustang S chemistry.
HEPES buffer (pH 7.0) was added to a final concentration of either 5 or 10 mM and applied to the plate. As expected the BSA ends up in the flow through indicating an inability to bind except for a small amount in 10 mM HEPES (negative values on the plot represent the concentrations recovered in the flow through) . The far right bars are positive controls with Mustang Q membrane. The data show the mean of n=8 samples.
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BSA Recovery with
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Figure 4
Lysozyme Recoveries with Mustang Q and S Membranes
Lysozyme is a highly charged (positive) protein that can be used as a control for IE protocol development since it is capable of tightly binding to Mustang S chemistry.
HEPES buffer was added to a final concentration of 10 mM and applied to either the
Mustang Q or Mustang S plate. Lysozyme ends up in the flow through of Mustang Q plate indicating an inability to bind (negative values on the plot represent the concentrations recovered in the flow through) and as expected the Lysozyme was bound and recovered at high efficiency from Mustang S plate. The data show the mean of n=8.
Lysozyme Recovery with
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©2004 Pall Corporation. Pall, , AcroPrep, Mustang, and Nanosep are registered trademarks of
Pall Corporation.
® indicates a trademark registered in the USA.
GN 04.0909 01/04
Figure 5
Duplicate Recoveries from Bovine Plasma HPLC Fractions on Mixed-Bed Mustang Q and S Multi-well Plates.
Independent extractions (two different weeks) from the bovine-plasma HPLC fractions were performed using plates sealed with both Mustang Q and Mustang S membranes in the same plate. The proteins were bound in 10 mM HEPES (final) and eluted in 1 mL 1N NaCl. Selected samples (20 µL from the 1 mL eluent) from the collection plate were electrophoresed on
PAGE, transferred and stained for visualization. BSA is seen in effluents 23 and 29 indicating reliable BSA recovery (est 90%).
Figure 6
Ultrafiltration Concentrated Extracted Bovine Plasma
Protein Fractions
Selected samples from an experimental run using the same conditions reported in Figure 5 were concentrated by ultrafiltration. The same fraction numbers from the 96-well collection plate were concentrated using a Nanosep ® 10K device and spun for 10 min at high speed. The resulting samples were recovered in 20 µL buffer and electrophoresed by PAGE. Reproducible patterns were detected (compare with Figure 5) at higher concentrations of the Gel.
Figure 7
Automation of Sample Preparation for Human Plasma
The (Initial Trace) below was generated while preparing the 150 µL Human Plasma sample for an experimental run using the same buffer and conditions reported for Figure 6. The Arrows indicate the start and end of the fraction collection. The graph below (Recovered Proteins) indicates the total protein recovery after elution in 1M NaCl as quantitated by the micro
BCA total protein assay on 10 µL of the respective eluent. Note the matching peaks for the recovered proteins for the BCA assay which is consistent with the initial trace.
Recovered Proteins
Initial Trace
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Figure 8
Human Plasma Fractions, Mixed Bed Q/S and Ultrafiltration
Selected samples from the experimental presented in Figure 7 was done using using the same buffer and processing condition reported previously with the exception that a TomTec Robotic filter station (Qaudra 3, TomTec, Hamdon, CT) was used for the liquid handling steps after collection. Based on BCA data, select fractions were concentrated using a Nanosep 10K device and spun for 10 min at high speed. The resulting samples were recovered in 20 µL buffer and electrophoresed by PAGE. The presence of the HSA bands clearly show the effectiveness of the automated recovery of
Human serum proteins from the HPLC fractions.
Figure 9
Trypsin Digestion of the HSA Fraction in Different Solvents
Human Serum Albumin (HSA)-containing fractions from HPLC elution (in 50% acetonitrile) and ion-exchange membrane elution (in 1M NaCl) were subject to overnight digestion with 0.5 µg sequencing-grade modified trypsin. As a control, 1 µg of HSA was trypisinized in 20 mM Ammonium Phosphate buffer, pH 8. The eluted/desalted samples (0.5 ul) were applied on a thin layer of dried matrix (alpha-cyano-hydroxy cinamic acid) pre-deposited on the MALDI target plate. The samples were dried and analyzed by Proteomic Analyzer
4700 MALDI mass spectrometer (ABI). The 1N NaCl sample gave an equivalent digestion to the control sample while the ACN (`50%) sample did not digest well.
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The AcroPrep ™ 96 filter plate is designed to fit most of the manual manifolds as well as robotic equipment. It has the following advantages:
• Polypropylene construction is chemically resistant and biologically inert
• Individually sealed membranes prevent lateral flow and crosstalk
• Rigid single piece construction
• Proprietary design minimizes solution/sample weeping
• Patented sealing technology allows the insertion of multiple layers of membrane
Mustang ™ chromatography’s membranes have been chemically modified with charged polymers that are crosslinked to the membrane’s pore surfaces, and this technology gives manufacturers several processing options. Mustang S membranes (negatively-charged) may be used to capture a wide variety of neutral to positively charged proteins. Alternatively, Mustang Q membranes
(positively-charged) are most effective at capturing neutral to negatively charged proteins. Since both elute almost all proteins at high salt (1M) the mixed bed configuration can be used to capture and elute virtually all of the proteins in a given population.
The combination of our unique sealing technology allowing multiple layers of membrane to be added to each well together with the high capacity of the Mustang membrane allows high throughput extractions to be a reality.
• Reverse phase HPLC fractions can be collected but not trypsinized flowing HIC chromatography. Since organic solvents can interfere with the digestion of proteins following HPLC, a buffer exchange is needed if digestion is needed for MALDI-TOF analysis.
• Ion exchange capture and elution can be effectively performed with organic-solvent containing solutions using a mixed-bed AcroPrep device where alternating layers of Mustang Q, and Mustang S membranes are sealed into an automation compatible filtration plate.
• The proteins recovered from the ion exchange plate can be further concentrated or desalted using ultrafiltration plates producing in ninety-six reproducible fractions fully capable of enzyme digestion and subsequent analysis.
• The use of the Pall Mustang ion exchange membranes together with the AcroPrep Plate platform allows proteomic analysis separating the proteins via chromatography followed by trypsin digestion and analysis on MALDI-TOF as an alternative or supplement to 2D-gel extraction.