- Email: [email protected]
or protein sequencing
Online Preparation Of Complex Biological Samples Prior To Analysis By HPLC, LC/MS And/Or Protein Sequencing Ken Stoney and Kerry Nugent Michrom BioResources, Inc., Auburn CA 95603
I. Introduction Although modem analytical techniques are very powerful for tracelevel characterization of proteins and peptides in complex biological samples, most samples require some degree of preparation prior to final analysis (1,2). The reason for this is that sample matrices and dilute samples generally interfere with the potency of analytical techniques. Operations such as concentration, desalting, buffer exchange and detergent removal which can correct matrix problems, are usually performed off line; this is often time consuming and can result in loss of the analytes of interest when working at low picomole levels (3). A series of micro trap cartridge columns have been developed which address the removal of several of the most common interfering substances found in biological matrices; these cartridges can be used in conjuction with HPLC analysis, or prior to Mass Spec, AAA or Protein Sequencing Analysis. Each type of trap cartridge has a chemistry which is uniquely suited to removal of a particular interfering substance. The first of these micro trap cartridges functions in salt removal and sample concentration. This micro trap cartridge can also be used to remove buffers and salts from biological samples, or perform a buffer exchange to a system more compatible with the final analytical technique. Mass Spectrometry is extremely sensitive to nonvolatile salts, which can cause instrumental problems, interfere with ionization of samples and make data interpretation much more difficult. Protein sequencers can also have difficulties with salts, especially phosphate, which can result TECHNIQUES IN PROTEIN CHEMISTRY VI Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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in a suppressed signal. For sample concentration, this trap cartridge uses reversed-phase HPLC chemistry to concentrate aqueous samples at the head of the trap, then releases the sample in a 10 ul volume when the cartridge is eluted with strong solvent. Volume reduction can be extremely important for such techniques as microbore/capillary HPLC and protein sequencing. With small ID HPLC columns running at lowflowrates (5-50^il/min), sample loading time can be as long as the separation time, in addition to adding large loop volumes to the gradient delay volume of the system. The use of multiple injections can get around sample volume problems, but adds significantly to the time involved for either procedure. Two different detergent removal cartridges have also been developed to help clean up biological samples prior to final analysis. The first of these removes anionic detergents such as Sodium Dodecyl Sulfate (SDS). SDS is widely used for solubilization of peptides and proteins, and is present in samples isolated from SDS-PAGE gels. With even trace levels of SDS present in the sample, chromatographic resolution and repeatability are compromised, and RP HPLC columns quickly become contaminated. Trace levels of SDS bound to peptides and proteins also makes mass spectral interpretation more difficult, and excess SDS can interfere with peptide and protein ionization and make it impossible to interpret the mass spectral data, especially when the analytes of interest are present at trace levels. SDS may also interfere with AAA and protein sequence analysis, and excess levels of SDS should be removed from samples prior to final analysis if accurate data is to be expected (4,5). The SDS removal cartridge uses a strong anion exchange chemistry to retain SDS molecules while proteins and peptides are released for analysis. The second detergent removal cartridge cleans up samples containing non ionic detergents (NID), such as Triton XI00, Tween 80, etc. NIDs are commonly used by protein chemists to help solubilize hydrophobic proteins. Like SDS, these detergents can interfere with both the chromatography and mass spectral interpretation for samples analyzed by LC/MS. They offer an even greater challenge than SDS, since they tend to be broad range mixtures of long chain surfactants that elute from a RP HPLC column over part or the entire range where proteins elute from the column. The non ionic detergent removal cartridge uses a mixed-bed ion-exchange chemistry and a multistep procedure to isolate proteins of interest from these potential interferences.
Online Sample Preparation
279
II. Materials and Methods Protein standards and detergents were obtained from Sigma Chemical Company, St. Louis, MO. Horse heart myoglobin from Sigma was digested with trypsin using a standard protocol from Promega. HPLC solvents were obtained from Burdick and Jackson and trifluoroacetic acid (TFA) was obtained from Pierce Chemical Company. All of the HPLC instrumentation, accessories, HPLC columns and micro trap cartridges are products of Michrom BioResources. All of the trap cartridges were used in an injection loop on the 10port valve built into the UMA. All of the peptide separations were run on a 1.0 X 150 mm column packed with Sju 300A Reliasil CI8 (Column Engineering, Ontario, CA) at 50 ul/min, using a 20 minute gradient from 565% Acetonitrile in water with 0.1% TFA. All of the protein separations were run on a 1.0 x 50 mm column packed with 8]LI 4000A PLRP-S (Polymer Labs, Amherst, MA) at 100 jul/min, using a 5 minute gradient from 5-65% Acetonitrile in water with 0.1% TFA. All of the HPLC separations were done using an Ultrafast Microprotein Analyzer (UMA) from Michrom BioResources.
III. Results and Discussion
A. Sample Concentration & De-Salting The top chromatogram in Figure 1 shows the separation of a myoglobin tryptic digest after 20 repetitive injections of 50 ul of 0.1 pmol/ jul dissolved in a 2M Urea solution. The large solvent front is due to the Urea which is not retained on the RPLC column. If this sample were run directly into an electrospray Mass Spectrometer, the large amount of Urea would greatly disturb the ion source, and could potentially plug the interface. Although the separation looks good, because all of the peptides were concentrated at the head of the column during the isocratic loading at 2% Acetonitrile in water with 0.1% TFA, what is not shown is the fact that loading time for this sample was 40 minutes, with each 50 jul injection wash having been washed out of the loop for two minutes prior to the next loading, and this procedure repeated 20 times. In the lower chromatogram in Figure 1, the same 1000 )ul sample has
280
Ken Stoney and Kerry Nugent
Figure 1. Concentration & removal of Urea from myoglobin tryptic digest prior to microbore LC/MS using a Michrom Peptide Trap Cartridge. Sample contains 100 pmol of digest dissolved in 1000 jul of 2 M Urea. Sample is run on a 1 x 150 mm RC-18 column.
been loaded in 2 minutes onto a peptide trap cartridge built into the injection loop of the HPLC; the cartridge is then rinsed with 100 jul of initial mobile phase (5% Acetonitrile in water with 0.1% TFA) to flush all of the Urea from the trap, prior to switching the sample on line to the analytical column for rapid gradient separation of the protein mixture. The large solvent front is absent from this lower trace, showing that the Urea has been selectively removed from the sample without loss of analyte. This rapid on-line desalting is accomplished by placing a Michrom protein or peptide trap cartridge in the injector loop, where samples containing salts can be loaded onto the trap; then the trap is rinsed with a volatile weak solvent (Solvent A from the HPLC) to insure complete removal of all the salts. The proteins and peptides of interest can then be immediately stepped off the trap cartridge with a strong solvent (Solvent B from the HPLC) into a few microliters of sample volume, or injected into a HPLC flow stream for further separation and / or analysis by LC/MS. This technique allows rapid concentration of samples from 20-10,000 \xl down to a 10 jul volume, and has been successfully employed to remove a wide range of salts and buffers (up to 8M) from a variety of protein and peptide samples.
Online Sample Preparation
281
Figure 2. On-line SDS removal from myoglobin tryptic digest prior to microbore HPLC, with/without SDS Removal Trap Cartridge, Sample contains 100 pmol of digest with/without 0.1% SDS. Sample is run on a 1 x 150 mm RC-18 column.
B. Anionic Detergent (SDS) Removal In Figure 2, the upper trace shows the separation of 100 picomoles of a myoglobin tryptic digest injected from a standard 20 jul loop without any SDS in the sample. The middle chromatogram shows the separation of the same 100 picomole myoglobin digest, but in a 0.1 % SDS solution, also injected from a standard 20 jul loop. The SDS binds to the peptides making them all more hydrophobic and more similar in overall polarity, thus resulting in the peaks being bunched up at the end of the chromatogram, with much worse overall resolution. The bottom chromatogram shows the same sample as the middle trace (in 0.1% SDS), but for this run, the loop was replaced with an micro SDS removal cartridge / loop system which was able to remove most of the SDS from the sample, such that the subsequent peptide separation was now very similar to that in the upper trace. The Michrom SDS removal cartridge can remove SDS at concentrations up to 1% (higher concentrations form micelles and trap analytes with the SDS micelle complex; these samples must be diluted below 1% prior to analysis), and can remove up to 1 mg of SDS from a single sample. When these cartridges are used in alO-port loop
282
Ken Stoney and Kerry Nugent
injector, the SDS can be removed while the peptides and proteins are trapped at the head of the RPLC column. The trap cartridge can then be switched out of line and back flushed (manually or automatically) with strong solvent during the HPLC run to completely remove the SDS from the cartridge without having it go through the HPLC column. This SDS cartridge also removes Coumassie Blue dye, an anionic stain commonly used in slab gel electrophoresis.
C Non Ionic Detergent Removal In Figure 3, the lower trace shows the separation of 200 ng of three standard proteins (insulin, lysozyme and alpha lactalbumin), without any detergent in the sample. The upper trace shows the same protein standard in a solution of 1.0% Triton X-100 detergent. Since Triton X-100 absorbs in the UV, a very large peak is seen for the detergent, but the peaks for the
Figure 3. On-line removal of Triton X-100 from proteins prior to microbore HPLC, with/ without NID Removal Trap Cartridge. Sample contains 200 ng of 3 protein standard with/ without Triton. Sample is run on a 1 x 50 mm PLRP-S column.
Online Sample Preparation
283
three standard proteins are completely obscured by the large detergent peak. The middle trace shows the results of running the same sample as in the upper trace (3 protein standard in 1.0 % Triton X-100), but using the non ionic detergent removal protocol. One can see that the middle and bottom traces are nearly identical, showing that the majority of the detergent has been removed from the sample by the non ionic detergent removal system. A multistep, automated procedure has been developed to selectively remove these non ionic interferences from proteins, using a non ionic detergent (NID) removal column in series with a protein trap cartridge, prior to separation on a RP HPLC column. Protein samples are loaded on to a mixed bed ion exchange precolumn (1x10mm) with 10% acetonitrile in 10 mM buffer (pH 5-8 depending on pi of protein) with the protein trap cartridge out of line in the valve loop. The protein(s) of interest are trapped on the precolumn (up to 1 mg of total protein), while the non ionic interferences are flushed to waste with the load solvent. The protein trap cartridge is then switched in line, and the proteins are eluted from the precolumn onto the trap cartridge with 10% acetonitrile in 2M NaCl buffer (pH 5 - 8). The salts are then washed out of the trap cartridge with 10% acetonitrile in 0.1 % TFA. Finally, the trap cartridge is returned in line to the RP HPLC column and the proteins are eluted, detergent free, through the RP HPLC column with the appropriate gradient conditions.
IV.
Conclusions
Complex biological samples often require some degree of sample preparation prior to final analysis by HPLC, LC/MS, Mass Spectrometry, AAA or Protein Sequencing due to sample matrix interferences. Although many off line procedures have been employed in the past to deal with problems of low concentration, high salt background and interferences from other additives such as detergents, these techniques can be time consuming and may result in significant loss of the analytes of interest when working at low levels. On-line sample preparation techniques, such as the trap cartridge systems described here, offer distinct advantages of minimizing prep time and maximizing sample recovery over other conventional sample preparation protocols. Use of the RP salt removal/concentration trap cartridges provides a rapid means for volume reduction, salt elimination and/or buffer exchange, since the entire procedure takes place in less than 5 minutes. With volumes
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Ken Stoney and Kerry Nugent
reduced from 10,000 jul down to 10 jul, samples can now be placed directly on to sequencing filters without further handling; volume reduction also avoids additional system volume in microbore HPLC. Removal of salts improves instrumental performance and data interpretation for mass spectrometry, AAA and protein sequence analysis. The SDS removal trap cartridges are able to remove up to 1 mg of SDS from a given sample. Use of these cartridges eliminates interference with reversed-phase separations and avoids column fouling which is commonly experienced when SDS is a part of the separation. Removal of SDS from biological samples also improves the accuracy of analysis by MS, AAA or sequencing. Non-ionic detergent (NID) removal cartridges used in the multistep procedure outline in this paper offer efficient cleanup, in addition to concentration of samples. Removal of NID's restores reversed-phase HPLC separation of proteins, as well as avoiding problems with mass spectral data interpretation. Future work is planned with these cartridges to see if they can be extended to other contaminants such as PolyEthylene Glycol (PEG), and a trap cartridge for removal of non ionic detergents from peptide samples is also being investigated.
References 1. 2. 3. 4.
5.
Atherton, D. (1989) In "Techniques in Protein Chemistry" (Hugli,T.E., ed.) 273283. Slattery, T.K. and Harkins, R.N. (1993) In "Techniques in Protein Chemistry IV" (Angeletti, R.H., ed.) 443-452. Nugent, K.D. and Nugent, P.W., (1990) Biochromatography 5(3), 142-148. Stone, K.L., LoPresti, M.B., Williams, N.D., Crawford, J.M., DeAngelis, R. and Williams, K.R. (1989) In "Techniques in Protein Chemistry" (Hugh, T.E., ed.) 377-391. Jeno, P., Scherer, Manning-Krieg, U. and Horst, M. (1993) Anal. Biochem. 215, 292-298.
I. Introduction Although modem analytical techniques are very powerful for tracelevel characterization of proteins and peptides in complex biological samples, most samples require some degree of preparation prior to final analysis (1,2). The reason for this is that sample matrices and dilute samples generally interfere with the potency of analytical techniques. Operations such as concentration, desalting, buffer exchange and detergent removal which can correct matrix problems, are usually performed off line; this is often time consuming and can result in loss of the analytes of interest when working at low picomole levels (3). A series of micro trap cartridge columns have been developed which address the removal of several of the most common interfering substances found in biological matrices; these cartridges can be used in conjuction with HPLC analysis, or prior to Mass Spec, AAA or Protein Sequencing Analysis. Each type of trap cartridge has a chemistry which is uniquely suited to removal of a particular interfering substance. The first of these micro trap cartridges functions in salt removal and sample concentration. This micro trap cartridge can also be used to remove buffers and salts from biological samples, or perform a buffer exchange to a system more compatible with the final analytical technique. Mass Spectrometry is extremely sensitive to nonvolatile salts, which can cause instrumental problems, interfere with ionization of samples and make data interpretation much more difficult. Protein sequencers can also have difficulties with salts, especially phosphate, which can result TECHNIQUES IN PROTEIN CHEMISTRY VI Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
277
278
Ken Stoney and Kerry Nugent
in a suppressed signal. For sample concentration, this trap cartridge uses reversed-phase HPLC chemistry to concentrate aqueous samples at the head of the trap, then releases the sample in a 10 ul volume when the cartridge is eluted with strong solvent. Volume reduction can be extremely important for such techniques as microbore/capillary HPLC and protein sequencing. With small ID HPLC columns running at lowflowrates (5-50^il/min), sample loading time can be as long as the separation time, in addition to adding large loop volumes to the gradient delay volume of the system. The use of multiple injections can get around sample volume problems, but adds significantly to the time involved for either procedure. Two different detergent removal cartridges have also been developed to help clean up biological samples prior to final analysis. The first of these removes anionic detergents such as Sodium Dodecyl Sulfate (SDS). SDS is widely used for solubilization of peptides and proteins, and is present in samples isolated from SDS-PAGE gels. With even trace levels of SDS present in the sample, chromatographic resolution and repeatability are compromised, and RP HPLC columns quickly become contaminated. Trace levels of SDS bound to peptides and proteins also makes mass spectral interpretation more difficult, and excess SDS can interfere with peptide and protein ionization and make it impossible to interpret the mass spectral data, especially when the analytes of interest are present at trace levels. SDS may also interfere with AAA and protein sequence analysis, and excess levels of SDS should be removed from samples prior to final analysis if accurate data is to be expected (4,5). The SDS removal cartridge uses a strong anion exchange chemistry to retain SDS molecules while proteins and peptides are released for analysis. The second detergent removal cartridge cleans up samples containing non ionic detergents (NID), such as Triton XI00, Tween 80, etc. NIDs are commonly used by protein chemists to help solubilize hydrophobic proteins. Like SDS, these detergents can interfere with both the chromatography and mass spectral interpretation for samples analyzed by LC/MS. They offer an even greater challenge than SDS, since they tend to be broad range mixtures of long chain surfactants that elute from a RP HPLC column over part or the entire range where proteins elute from the column. The non ionic detergent removal cartridge uses a mixed-bed ion-exchange chemistry and a multistep procedure to isolate proteins of interest from these potential interferences.
Online Sample Preparation
279
II. Materials and Methods Protein standards and detergents were obtained from Sigma Chemical Company, St. Louis, MO. Horse heart myoglobin from Sigma was digested with trypsin using a standard protocol from Promega. HPLC solvents were obtained from Burdick and Jackson and trifluoroacetic acid (TFA) was obtained from Pierce Chemical Company. All of the HPLC instrumentation, accessories, HPLC columns and micro trap cartridges are products of Michrom BioResources. All of the trap cartridges were used in an injection loop on the 10port valve built into the UMA. All of the peptide separations were run on a 1.0 X 150 mm column packed with Sju 300A Reliasil CI8 (Column Engineering, Ontario, CA) at 50 ul/min, using a 20 minute gradient from 565% Acetonitrile in water with 0.1% TFA. All of the protein separations were run on a 1.0 x 50 mm column packed with 8]LI 4000A PLRP-S (Polymer Labs, Amherst, MA) at 100 jul/min, using a 5 minute gradient from 5-65% Acetonitrile in water with 0.1% TFA. All of the HPLC separations were done using an Ultrafast Microprotein Analyzer (UMA) from Michrom BioResources.
III. Results and Discussion
A. Sample Concentration & De-Salting The top chromatogram in Figure 1 shows the separation of a myoglobin tryptic digest after 20 repetitive injections of 50 ul of 0.1 pmol/ jul dissolved in a 2M Urea solution. The large solvent front is due to the Urea which is not retained on the RPLC column. If this sample were run directly into an electrospray Mass Spectrometer, the large amount of Urea would greatly disturb the ion source, and could potentially plug the interface. Although the separation looks good, because all of the peptides were concentrated at the head of the column during the isocratic loading at 2% Acetonitrile in water with 0.1% TFA, what is not shown is the fact that loading time for this sample was 40 minutes, with each 50 jul injection wash having been washed out of the loop for two minutes prior to the next loading, and this procedure repeated 20 times. In the lower chromatogram in Figure 1, the same 1000 )ul sample has
280
Ken Stoney and Kerry Nugent
Figure 1. Concentration & removal of Urea from myoglobin tryptic digest prior to microbore LC/MS using a Michrom Peptide Trap Cartridge. Sample contains 100 pmol of digest dissolved in 1000 jul of 2 M Urea. Sample is run on a 1 x 150 mm RC-18 column.
been loaded in 2 minutes onto a peptide trap cartridge built into the injection loop of the HPLC; the cartridge is then rinsed with 100 jul of initial mobile phase (5% Acetonitrile in water with 0.1% TFA) to flush all of the Urea from the trap, prior to switching the sample on line to the analytical column for rapid gradient separation of the protein mixture. The large solvent front is absent from this lower trace, showing that the Urea has been selectively removed from the sample without loss of analyte. This rapid on-line desalting is accomplished by placing a Michrom protein or peptide trap cartridge in the injector loop, where samples containing salts can be loaded onto the trap; then the trap is rinsed with a volatile weak solvent (Solvent A from the HPLC) to insure complete removal of all the salts. The proteins and peptides of interest can then be immediately stepped off the trap cartridge with a strong solvent (Solvent B from the HPLC) into a few microliters of sample volume, or injected into a HPLC flow stream for further separation and / or analysis by LC/MS. This technique allows rapid concentration of samples from 20-10,000 \xl down to a 10 jul volume, and has been successfully employed to remove a wide range of salts and buffers (up to 8M) from a variety of protein and peptide samples.
Online Sample Preparation
281
Figure 2. On-line SDS removal from myoglobin tryptic digest prior to microbore HPLC, with/without SDS Removal Trap Cartridge, Sample contains 100 pmol of digest with/without 0.1% SDS. Sample is run on a 1 x 150 mm RC-18 column.
B. Anionic Detergent (SDS) Removal In Figure 2, the upper trace shows the separation of 100 picomoles of a myoglobin tryptic digest injected from a standard 20 jul loop without any SDS in the sample. The middle chromatogram shows the separation of the same 100 picomole myoglobin digest, but in a 0.1 % SDS solution, also injected from a standard 20 jul loop. The SDS binds to the peptides making them all more hydrophobic and more similar in overall polarity, thus resulting in the peaks being bunched up at the end of the chromatogram, with much worse overall resolution. The bottom chromatogram shows the same sample as the middle trace (in 0.1% SDS), but for this run, the loop was replaced with an micro SDS removal cartridge / loop system which was able to remove most of the SDS from the sample, such that the subsequent peptide separation was now very similar to that in the upper trace. The Michrom SDS removal cartridge can remove SDS at concentrations up to 1% (higher concentrations form micelles and trap analytes with the SDS micelle complex; these samples must be diluted below 1% prior to analysis), and can remove up to 1 mg of SDS from a single sample. When these cartridges are used in alO-port loop
282
Ken Stoney and Kerry Nugent
injector, the SDS can be removed while the peptides and proteins are trapped at the head of the RPLC column. The trap cartridge can then be switched out of line and back flushed (manually or automatically) with strong solvent during the HPLC run to completely remove the SDS from the cartridge without having it go through the HPLC column. This SDS cartridge also removes Coumassie Blue dye, an anionic stain commonly used in slab gel electrophoresis.
C Non Ionic Detergent Removal In Figure 3, the lower trace shows the separation of 200 ng of three standard proteins (insulin, lysozyme and alpha lactalbumin), without any detergent in the sample. The upper trace shows the same protein standard in a solution of 1.0% Triton X-100 detergent. Since Triton X-100 absorbs in the UV, a very large peak is seen for the detergent, but the peaks for the
Figure 3. On-line removal of Triton X-100 from proteins prior to microbore HPLC, with/ without NID Removal Trap Cartridge. Sample contains 200 ng of 3 protein standard with/ without Triton. Sample is run on a 1 x 50 mm PLRP-S column.
Online Sample Preparation
283
three standard proteins are completely obscured by the large detergent peak. The middle trace shows the results of running the same sample as in the upper trace (3 protein standard in 1.0 % Triton X-100), but using the non ionic detergent removal protocol. One can see that the middle and bottom traces are nearly identical, showing that the majority of the detergent has been removed from the sample by the non ionic detergent removal system. A multistep, automated procedure has been developed to selectively remove these non ionic interferences from proteins, using a non ionic detergent (NID) removal column in series with a protein trap cartridge, prior to separation on a RP HPLC column. Protein samples are loaded on to a mixed bed ion exchange precolumn (1x10mm) with 10% acetonitrile in 10 mM buffer (pH 5-8 depending on pi of protein) with the protein trap cartridge out of line in the valve loop. The protein(s) of interest are trapped on the precolumn (up to 1 mg of total protein), while the non ionic interferences are flushed to waste with the load solvent. The protein trap cartridge is then switched in line, and the proteins are eluted from the precolumn onto the trap cartridge with 10% acetonitrile in 2M NaCl buffer (pH 5 - 8). The salts are then washed out of the trap cartridge with 10% acetonitrile in 0.1 % TFA. Finally, the trap cartridge is returned in line to the RP HPLC column and the proteins are eluted, detergent free, through the RP HPLC column with the appropriate gradient conditions.
IV.
Conclusions
Complex biological samples often require some degree of sample preparation prior to final analysis by HPLC, LC/MS, Mass Spectrometry, AAA or Protein Sequencing due to sample matrix interferences. Although many off line procedures have been employed in the past to deal with problems of low concentration, high salt background and interferences from other additives such as detergents, these techniques can be time consuming and may result in significant loss of the analytes of interest when working at low levels. On-line sample preparation techniques, such as the trap cartridge systems described here, offer distinct advantages of minimizing prep time and maximizing sample recovery over other conventional sample preparation protocols. Use of the RP salt removal/concentration trap cartridges provides a rapid means for volume reduction, salt elimination and/or buffer exchange, since the entire procedure takes place in less than 5 minutes. With volumes
284
Ken Stoney and Kerry Nugent
reduced from 10,000 jul down to 10 jul, samples can now be placed directly on to sequencing filters without further handling; volume reduction also avoids additional system volume in microbore HPLC. Removal of salts improves instrumental performance and data interpretation for mass spectrometry, AAA and protein sequence analysis. The SDS removal trap cartridges are able to remove up to 1 mg of SDS from a given sample. Use of these cartridges eliminates interference with reversed-phase separations and avoids column fouling which is commonly experienced when SDS is a part of the separation. Removal of SDS from biological samples also improves the accuracy of analysis by MS, AAA or sequencing. Non-ionic detergent (NID) removal cartridges used in the multistep procedure outline in this paper offer efficient cleanup, in addition to concentration of samples. Removal of NID's restores reversed-phase HPLC separation of proteins, as well as avoiding problems with mass spectral data interpretation. Future work is planned with these cartridges to see if they can be extended to other contaminants such as PolyEthylene Glycol (PEG), and a trap cartridge for removal of non ionic detergents from peptide samples is also being investigated.
References 1. 2. 3. 4.
5.
Atherton, D. (1989) In "Techniques in Protein Chemistry" (Hugli,T.E., ed.) 273283. Slattery, T.K. and Harkins, R.N. (1993) In "Techniques in Protein Chemistry IV" (Angeletti, R.H., ed.) 443-452. Nugent, K.D. and Nugent, P.W., (1990) Biochromatography 5(3), 142-148. Stone, K.L., LoPresti, M.B., Williams, N.D., Crawford, J.M., DeAngelis, R. and Williams, K.R. (1989) In "Techniques in Protein Chemistry" (Hugh, T.E., ed.) 377-391. Jeno, P., Scherer, Manning-Krieg, U. and Horst, M. (1993) Anal. Biochem. 215, 292-298.