Automated On-Line SDS Removal from Diminutive Protein Samples Prior to Capillary HPLC Application Note 221

Transcription

1 Automated On-Line SDS Removal from Diminutive Protein Samples Prior to Capillary HPLC Application Note 221 Joan Stevens, Ph.D.; Luke Roenneburg; Tim Hegeman (Gilson, Inc.) Introduction Removing detergent from small amounts of protein and peptide samples can be a formidable problem, especially if a reverse-phase HPLC (RP HPLC) separation is used for analysis or purification. The presence of ionic detergents, such as SDS, is known to destroy any chromatographic resolution on RP HPLC even in very low concentrations making mass spectral (MS) analysis almost impossible. Some of the issues include: Ion signal derived from detergent often suppresses ion signal of peptides and proteins present in the same fraction Detergents often elute over the whole gradient of the chromatogram, contaminating every fraction with detergent Problem arises whether the fractions were collected off line and analyzed by MS or continuously delivered into MS during LC-MS analysis Detergents often do not have one distinct mass that could be filtered out during MS analysis SDS removal is accomplished through a combination of anion exchange and hydrophilic interaction. SDS is retained on the trapping column, while peptides proceed to the reverse-phase column. Using a trifluoroacetic acid (TFA)-containing mobile phase, peptides are eluted with a gradient of organic solvent. When the organic level exceeds 70%, at least 95% of the SDS desorbs and is eluted to waste. This can be accomplished by switching the SDS trapping column off line and washing it with a high organic solution at low ph. The trapping column and capillary column can then be re-equilibrated for the next sample. Materials & Methods System Components Gilson 350 Micro Pumps (2), equipped with: nano mixer assembly Gilson 235 Autoinjector, equipped with: 1-µL internal loop Gilson VALVEMATE Valve Actuator, equipped with: 7610 Rheodyne valve (10-port, 2-position) Gilson 155 UV/VIS Dual-wavelength Detector, equipped with: capillary flow cell (35 nl x 8 mm, 0.01 AUFS, 210 nm) Gilson 307 HPLC Pump, equipped with: 5-mL pump head For Fraction Collection onto MALDI Plates: Gilson 223 Fraction Collector, equipped with: spring-loaded probe 402 Matrix Pump, equipped with: matrix valve January 2004 Page 1 of

2 Figure 1: System Hardware Configuration without Fraction Collection Figure 2: System Hardware Configuration with Fraction Collection The micro mixing tee sits atop the spring-loaded probe on the 223 Fraction Collector. The outlet of the detector is brought into the top of the tee. The Matrix Solution is continuously pumped into the tee by the dilutor pump. Figure 3: Representation of the Micro Mixing Tee January 2004 Page 2 of

3 Photo 1: Collection of Fractions Directly onto the MALDI Plate Insert represents the samples with matrix collected on the MALDI plate at various volumes. SDS Trapping Column Figure 4: SDS Trapping Column Diagram The SDS trapping column is a strong anion exchange column with a silica support; polymeric support is also available. (SAX-Reliasol, 40 µm, 1 µl 4 µg capacity, Optimize Technologies) Controlling Software The software allows complete control over the system, including on-line fraction collection directly onto a MALDI plate for analysis via MALDI-TOF MS. Multiple collection windows are available, each with their own time collection capability. The pumping of sample solution across the trap bed should result in the selective binding of SDS to the January 2004 Page 3 of

4 trap. The protein/peptides should pass through the trap unretained and can be sent for immediate analysis or subjected to further on-line purification steps. The SDS trapping column is switched off line automatically through the software for washing and eluting the trapped SDS, and then switched back into the system for equilibration prior to the next sample. Capillary HPLC Conditions and Methodology Mobile Phase Gradient: A = water (0.05% TFA)/B = ACN (0.05% TFA) % A/2% B % A/2% B to 70% A/30% B % A/30% B % A/30% B to 5% A/95% B % A/95% B % A/95% B to 98% A/2% B % A/2% B Flow Rate: 5 µl/min Sample Injected: 1 µl of BSA tryptic digest, with or without SDS addition HPLC Pump: 10 µl/min, 5% water (0.1% TFA)/95% ACN (0.1% TFA), ph 3.0 Results Chromatogram 1 Chromatogram 2 Chromatograms 1 & 2: BSA Tryptic Digest For a 1-µL injection, similar results are shown for the BSA digest without the SDS trapping column (chromatogram #1) or with the SDS trapping column (chromatogram #2). January 2004 Page 4 of

5 Chromatogram 3 Chromatogram 4 Chromatograms 3 & 4: Detrimental Effects of SDS on the Chromatography for the BSA Tryptic Digest SDS greatly interferes with eluting peptides. As the percentage of SDS increase, so does its degradation of the chromatography (as is indicated in the above chromatograms). Chromatogram 5 Chromatogram 6 Chromatograms 5 & 6: Effectiveness of the SDS Trapping Column to Eliminate SDS from the Peptides The SDS trapping column is taken off line at the end of the elution of the peptides. It is washed with 95% ACN/water (0.1% TFA) at 10 µl/ min for about 5 minutes (while the capillary column is also washed). The SDS trapping column is switched back in line, and both columns are brought back to initial conditions to await the next sample. January 2004 Page 5 of

6 Conclusion The use of a simple ion exchange trap provided the removal of SDS from the BSA tryptic digest, which allowed the sample to be analyzed. The use of a silica support for the anion exchanger offers a stable option at a ph of 3 or above. (A polymeric support is also available for a ph below 2.) The anion exchange trap will have a finite capacity for SDS trap and must be regenerated before the capacity is exceeded. SDS can be removed from the trap bed using a mobile phase that is both highly organic and acidic a combination that should ensure interruption of the interaction between the detergent and the anion exchange bed (e.g., ph below 4 and an organic content above 90%). The addition of the SDS trap adds no additional time to the analysis and offers tremendous advantages for the analysis of peptides/proteins associated with SDS. The use of a SDS trapping column greatly enhanced the analysis of the BSA tryptic digest. Without the SDS trapping column (as shown in chromatograms 3 and 4), the resolving power of the capillary HPLC column was masked by the SDS bleeding through the column with the peptides of interest. SDS not only inhibits the analysis of peptides and proteins via HPLC, it also renders MS useless because of its various masses and ion suppression. The addition of a SDS trapping column solved the detergent issue. Analysis of the BSA tryptic digest was restored without any additional time added onto the analysis. Regeneration of the SDS trapping column was accomplished off line by a switching valve, which introduced a high percentage of organic at a low ph. The SDS trapping column was then brought back on line and equilibrated with the capillary column prior to the next sample. The SDS trapping column offers a highly effective alternative to other off-line detergent removal methods. Gilson, Inc. World Headquarters 3000 W. Beltline Hwy., P.O. Box , Middleton, WI USA Telephone: (1) or (1) Fax: (1) Gilson S.A.S. 19, avenue des Entrepreneurs, BP 145, F VILLIERS LE BEL France Telephone: (33-1) Fax: (33-1) sales@gilson.com, service@gilson.com, training@gilson.com January 2004 Page 6 of