187,76-83
ANALYTICALBIOCHEMISTRY
Sample Centrifugation
(1990)
onto Membranes for Sequencing
Donald Sheer Applied
Biosystems
Received
October
Inc., 850 Lincoln
Center Driue, Foster City, California
16,1989
This paper presents a new method for adsorption of proteins in solution onto a polyvinylidene diflouride (PVDF) membrane using centrifugation. The technique uses a low molecular weight cut-off membrane (LMW) placed underneath a PVDF membrane. The paired membranes are placed in a receptacle which in turn fits into a microcentrifuge tube. During sample centrifugation, the LMW acts to increase the amount of protein that is concentrated and adsorbed onto the hydrophobic surface of the PVDF membrane. By alternating between two receptacle sizes, this method can accomodate large (>lO pg) and small (~10 pg) amounts of sample. This paper demonstrates sample recovery for a variety of proteins as quantitated by radioactivity and amino acid analysis after centrifugation onto PVDF. Amino acid and sequence analysis results demonstrate the efficiency with which interfering buffers and sodium dodecyl sulfate are removed as a result of sample centrifugation and washing. Finally, we demonstrate the utility of this technique with samples in the low picomole range to obtain useful sequence information following electrophoretic isolation of cyanogen bromide fragments purified by high performance electrophoresis chromatography. o 1990 Academic PWS. IN.
N-terminal sequences of proteins or peptides have been determined on starting amounts as low as 10 pmol using gas phase sequencing methodology with on-line phenylthiohydantoin (PTH)l amino acid analysis. Since many proteins have blocked N-termini, it is often necessary to enzymatically or chemically cleave them and isolate the resulting fragments prior to analysis. The availability and implementation of techniques to perform such work at submicrogram levels has become a ratelimiting step in protein sequencing. ’ Abbreviations used: PTH, phenylthiohydantoin; SDS, sodium dodecyl sulfate; PVDF, polyvinylidene difluoride; LMW, low molecular weight cut-off membrane; DI, deionized; TPI, triosephosphate isomerase; CPP, carboxypeptidase P. 76
94404
There are two requirements for obtaining such data from an unknown sample. The first is that the sample be relatively “free” of other proteinaceous contamination to assure that any sequence data generated can be attributed to the protein of interest. A second requirement is that the sample be free of chemicals and reagents which would interfere with the sequencing reactions. For example, amino groups from buffers containing Trisglycine or Tris-tricine will react with phenylisothiocyanate and give rise to “contaminant” peaks during HPLC analysis. Samples with contaminating detergents such as sodium dodecyl sulfate (SDS) or Triton X-100 can cause bubbling in automated sequencer lines which effects reagent and solvent flows in the instrument. Included among the sample clean-up procedures that have evolved over the years to remove interfering materials are gel filtration, dialysis, precipitation, and reverse-phase desalting. Each has its utility depending on the circumstances and the quantity of material available, and each has limitations when dealing with picomole amounts of material. More recently, polyvinylidene difluoride (PVDF) membranes have been used for electroblotting followed by direct sequencing (1,2). Although this membrane has rapidly emerged as one of the preferred substrates for electroblotting and sequencing, several problems can cause low recoveries or variable results. Those problems commonly encountered during electroblotting or sample adsorption onto PVDF include variability in electrotransfer for different proteins, long times (up to 24 h) required for adsorption of proteins from solution onto PVDF, variable results in recovery due to protein selectivity, and staining and destaining methods which decrease sequencing yields (3). The procedure described in this bulletin circumvents many of these problems by centrifuging dissolved samples through a membrane, i.e., PVDF, and then subsequently washing the membrane to remove contaminants. Samples can then be used for sequencing and amino acid analysis, or for fragmentation (by chemical or enzymatic methods) and subsequent isolation and analysis. 0003-2697/90$3.00
Copyright All rights
0 1990 by Academic
of reproduction
in any form
Press, Inc. reserved.
CENTRIFUGATION
MATERIALS
AND
TECHNIQUE
FOR
PROTEIN
‘II’----___ 0II
METHODS
All electrophoretic separations were carried out on an Applied Biosystems (ABI) Model 230A HPEC system using the Tris-glycine or Tris-tricine buffer systems (45); Immobilon P membrane (Millipore) was used in all experiments. Amino acid analysis was performed with an ABI Model 420A amino acid analyzer with an online ABI Model 130A separation module and peptide sequencing on an ABI Model 477A pulsed liquid phase sequencer with online Model 120A PTH analyzer. Processing of data was performed using the ABI Model 610A data analysis system. ABI reagents and solvents were used when available, otherwise the highest purity grade commonly available. The methylated-14C-labeled proteins were purchased from New England Nuclear (cytochrome c, &lactoglobulin, ovalbumin, methemoglobin, casein, bovine serum albumin, phosphorylase B, and myosin) or Amersham (insulin and aprotinin). Removal of 14Clabel by gel filtration (G25 Sepahadex, Pharmacia) was performed prior to sample centrifugation for the respective proteins. ,&Lactoglobulin (ABI) and cytochrome c (Sigma) were used to dilute the appropriate gel filtered methylated-14C-labeled protein and used as described in text.
77
ADSORPTION
- - Reservoir
--__
- - - - Sample
cap
reservoir
II
Protein Binding Membrane
@id ‘--_ - support
base
Application and Desalting by Centrifugation Adsorption of proteins in solution onto PVDF by centrifugation as an alternative sample loading and sample cleanup procedure is described below. Two procedures are described for large (>lO pg) or small (
ANALYTICALBIOCHEMISTRY
Sample Centrifugation
(1990)
onto Membranes for Sequencing
Donald Sheer Applied
Biosystems
Received
October
Inc., 850 Lincoln
Center Driue, Foster City, California
16,1989
This paper presents a new method for adsorption of proteins in solution onto a polyvinylidene diflouride (PVDF) membrane using centrifugation. The technique uses a low molecular weight cut-off membrane (LMW) placed underneath a PVDF membrane. The paired membranes are placed in a receptacle which in turn fits into a microcentrifuge tube. During sample centrifugation, the LMW acts to increase the amount of protein that is concentrated and adsorbed onto the hydrophobic surface of the PVDF membrane. By alternating between two receptacle sizes, this method can accomodate large (>lO pg) and small (~10 pg) amounts of sample. This paper demonstrates sample recovery for a variety of proteins as quantitated by radioactivity and amino acid analysis after centrifugation onto PVDF. Amino acid and sequence analysis results demonstrate the efficiency with which interfering buffers and sodium dodecyl sulfate are removed as a result of sample centrifugation and washing. Finally, we demonstrate the utility of this technique with samples in the low picomole range to obtain useful sequence information following electrophoretic isolation of cyanogen bromide fragments purified by high performance electrophoresis chromatography. o 1990 Academic PWS. IN.
N-terminal sequences of proteins or peptides have been determined on starting amounts as low as 10 pmol using gas phase sequencing methodology with on-line phenylthiohydantoin (PTH)l amino acid analysis. Since many proteins have blocked N-termini, it is often necessary to enzymatically or chemically cleave them and isolate the resulting fragments prior to analysis. The availability and implementation of techniques to perform such work at submicrogram levels has become a ratelimiting step in protein sequencing. ’ Abbreviations used: PTH, phenylthiohydantoin; SDS, sodium dodecyl sulfate; PVDF, polyvinylidene difluoride; LMW, low molecular weight cut-off membrane; DI, deionized; TPI, triosephosphate isomerase; CPP, carboxypeptidase P. 76
94404
There are two requirements for obtaining such data from an unknown sample. The first is that the sample be relatively “free” of other proteinaceous contamination to assure that any sequence data generated can be attributed to the protein of interest. A second requirement is that the sample be free of chemicals and reagents which would interfere with the sequencing reactions. For example, amino groups from buffers containing Trisglycine or Tris-tricine will react with phenylisothiocyanate and give rise to “contaminant” peaks during HPLC analysis. Samples with contaminating detergents such as sodium dodecyl sulfate (SDS) or Triton X-100 can cause bubbling in automated sequencer lines which effects reagent and solvent flows in the instrument. Included among the sample clean-up procedures that have evolved over the years to remove interfering materials are gel filtration, dialysis, precipitation, and reverse-phase desalting. Each has its utility depending on the circumstances and the quantity of material available, and each has limitations when dealing with picomole amounts of material. More recently, polyvinylidene difluoride (PVDF) membranes have been used for electroblotting followed by direct sequencing (1,2). Although this membrane has rapidly emerged as one of the preferred substrates for electroblotting and sequencing, several problems can cause low recoveries or variable results. Those problems commonly encountered during electroblotting or sample adsorption onto PVDF include variability in electrotransfer for different proteins, long times (up to 24 h) required for adsorption of proteins from solution onto PVDF, variable results in recovery due to protein selectivity, and staining and destaining methods which decrease sequencing yields (3). The procedure described in this bulletin circumvents many of these problems by centrifuging dissolved samples through a membrane, i.e., PVDF, and then subsequently washing the membrane to remove contaminants. Samples can then be used for sequencing and amino acid analysis, or for fragmentation (by chemical or enzymatic methods) and subsequent isolation and analysis. 0003-2697/90$3.00
Copyright All rights
0 1990 by Academic
of reproduction
in any form
Press, Inc. reserved.
CENTRIFUGATION
MATERIALS
AND
TECHNIQUE
FOR
PROTEIN
‘II’----___ 0II
METHODS
All electrophoretic separations were carried out on an Applied Biosystems (ABI) Model 230A HPEC system using the Tris-glycine or Tris-tricine buffer systems (45); Immobilon P membrane (Millipore) was used in all experiments. Amino acid analysis was performed with an ABI Model 420A amino acid analyzer with an online ABI Model 130A separation module and peptide sequencing on an ABI Model 477A pulsed liquid phase sequencer with online Model 120A PTH analyzer. Processing of data was performed using the ABI Model 610A data analysis system. ABI reagents and solvents were used when available, otherwise the highest purity grade commonly available. The methylated-14C-labeled proteins were purchased from New England Nuclear (cytochrome c, &lactoglobulin, ovalbumin, methemoglobin, casein, bovine serum albumin, phosphorylase B, and myosin) or Amersham (insulin and aprotinin). Removal of 14Clabel by gel filtration (G25 Sepahadex, Pharmacia) was performed prior to sample centrifugation for the respective proteins. ,&Lactoglobulin (ABI) and cytochrome c (Sigma) were used to dilute the appropriate gel filtered methylated-14C-labeled protein and used as described in text.
77
ADSORPTION
- - Reservoir
--__
- - - - Sample
cap
reservoir
II
Protein Binding Membrane
@id ‘--_ - support
base
Application and Desalting by Centrifugation Adsorption of proteins in solution onto PVDF by centrifugation as an alternative sample loading and sample cleanup procedure is described below. Two procedures are described for large (>lO pg) or small (
