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Separation and Analysis of Peptides at High pH Using RP-HPLC/ESI-MS

Separation and Analysis of Peptides at High pH Using RP-HPLC/ESI-MS. Barry E. Boyes 4th Symposium on the Analysis of Well Characterized Biotechnology Pharmaceuticals San Francisco, CA January 9-12, 2000. Introduction/Overview.

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Separation and Analysis of Peptides at High pH Using RP-HPLC/ESI-MS

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  1. Separation and Analysis of Peptides at High pH Using RP-HPLC/ESI-MS Barry E. Boyes 4th Symposium on the Analysis of Well Characterized Biotechnology Pharmaceuticals San Francisco, CA January 9-12, 2000

  2. Introduction/Overview Reversed-phase HPLC (RP-HPLC) separations combined with on-line mass spectrometry solves a variety of problems of biopharmaceutical and biochemical interest. Electrospray ionization mass spectrometry (ESI-MS) is readily coupled to RP-HPLC systems, and has become the most common method for interfacing mass analysis with high resolution separations. Analyses of protein or peptide mixtures by RP-HPLC are often conducted using gradient elution of organic solvent at low pH, where peak shapes, peak capacity, and recoveries are generally superior. In particular, trifluoroacetic acid (TFA) and other perfluorinated organic acids are excellent reagents for modifying the mobile phase, possessing good UV transparency, ion-pairing properties, protein and peptide solubilizing properties, and high volatility. TFA is generally used in the range of 0.02-0.2% in the mobile phase, in order to maintain the best peak shapes for peptides and proteins, with higher mass proteins requiring greater amounts of the acid. Although TFA is a highly useful reagent for RP-HPLC separations of peptides and proteins, it’s use in ESI-MS is problematic. The strong association of TFA with basic functional groups (N-terminus and amino acid side chains) results in suppression of positive ion formation during the electrospray process, limiting the utility of ESI-MS in sample-limited applications. In order to improve RP-HPLC/ESI-MS detection, the easily available choices are limited to decreasing the column internal diameter, which increases the complexity of the instrumentation, or modifying the chemistry of the ESI process. Based on results from infusion ESI-MS experiments (eg., Kelly et al., 1992, and see below), it has been known for some time that positive ion mass spectra can be obtained for amino acids and proteins at high pH, using ammonium hydroxide solution. There is a paucity of information available on the relative detection sensitivity of high versus low pH ESI-MS of peptides or proteins. Although it is possible that there may be advantages to conducting peptide separations at intermediate or elevated pH, the use of silica based materials has been limited, due to poor stability at intermediate or high pH. Recently, Kirkland et al. (1998), have reported the development of new highly-stable silica-based column packing materials. These materials use a unique bidentate ligand structure (see below) which aids in protecting the silica particle from dissolution. The combination of the bidentate silane surface modification with exhaustive end-capping results in a material with extreme resistance to dissolution at high pH. In the present study, a variety of peptide mixtures were examined using low, intermediate and high pH mobile phases, in order to compare chromatographic performance over the full range of pH. For comparison with TFA, ammonium bicarbonate and ammonium hydroxide were chosen, due to their high UV transmission and compatibility with the ESI-MS interface. The use of ESI-MS detection of polypeptides in ammonium hydroxide is compared to acetic acid and TFA, using flow injection analysis for apoMyoglobin, and RP-HPLC/ESI-MS for several peptide mixtures.

  3. C18 C18 S i S i O O Silica Support Zorbax® Extend C18 Bidentate Silane Surface Aging of Zorbax Extend-C18 (Bidentate-C18) Column in Ammonia at pH 10.5 Conditions: 4.6 x 150 mm; 80% Methanol / 20% 20 mM NH4OH, pH 10.5; 1.5 mL / min. Aging at 24°C; Tests at 40°C • During extended continuous purging of the Zorbax Extend C18 column at high pH, no significant chromatographic changes were observed, based on either retention or column efficiency measurements.

  4. 128 pmol 32 pmol 8 pmol 2 pmol 0 pmol 8E8 6E8 4E8 2E8 0 min 0 2 4 6 8 min 10 0 2 4 6 8 10 min 0 2 4 6 8 10 Experimental Mass Spectrometry System: HP1100 LC-MSD, ESI Interface HP Chemstation Data Processing Acquisition Conditions: Scan Range as Indicated - Peak width 0.07min. Peptides: 0.1 amu steps; Proteins: 0.15 amu steps 0.3 amu Gaussian Mass Filter MS Setting: Fixed Fragmentor Voltage, as Indicated (Vf) Electrospray Setting: Vcap = 4500 or 5000 V, as Indicated for positive ions = 4000 V for negative ions Nebulizing Gas- 35-45 p.s.i., as Indicated, Drying Gas- 300 or 325°C, 11 or 12LPM HPLC System: HP1100 LC with the Binary Pump, Temperature Controlled Column Compartment, Autosampler (10°C), and Diode Array Detector (micro flow cell, 6 mm, 1.7 µL) Chromatographic Conditions: Flow rate- 0.20 or 0.25 mL/min, Column Temp.- as Indicated Absorbance Detection: 8 nm slit width 210 nm, 10 nm BW, Ref. 400 nm, 100 nm BW HPLC Columns: All of the separations were conducted using 2.1 mm ID x 15 cm columns. Comparisons of separations at high versus low pH used the Zorbax Extend-C18 (Bidentate C18) column or a prototype 300Å pore size version of the same (Zorbax 300 Extend-C18). This columns uses a unique, patented bidentate C18 propylene-bridged bifunctional silane bonded-phase, which is multiply end-capped. This new column has been demonstrated by Kirkland et al. (1998) to withstand operation at extremes of pH for extended periods of time. Materials: The 3-and 4-residue peptide samples were obtained from Bachem Biosciences (Philadelphia PA). ApoMyoglobin, Angiotensin peptides, bovine Calmodulin (CaM), Lysozyme, bovine Insulin and oxidized B-chain Insulin (InsBox) were obtained from Sigma-Aldrich (St. Louis MO). The porcine intestinal 9k Calcium Binding Protein (Calbindin-D9k, CaD9) was obtained from Calbiochem (La Jolla CA). Amyloid ß-peptides (1-38), (1-40) and (1-42) were supplied by Dr. Anita Hong at AnaSpec (San Jose, CA). The Angiotensin peptides were obtained from Sigma-Aldrich (St. Louis MO). TFA was from Pierce Chemical (Rockford IL), and ammonium hydroxide (28% NH3 in water) was from Aldrich. Flow Injection Analysis of Equine apoMyoglobin: Effect of Solution Conditions on ESI-MS Conditions: 10 µL sample in mobile phase, 0.25 mL/min, Vf- 80V, Vcap- 5 kV, N2- 11L/min, 325°C, 40 psi 1% HOAc in 50% AcN 10 mM NH4OHin 50% AcN 0.1% TFA in 50% AcN TIC Intensity (300- 2500 m/z) • FIA/ESI-MS results for apoMyoglobin (MW 16951) show high Total Ion Chromatogram (TIC) intensity for NH4OH solutions, comparable to HOAc, and about 6-fold better than obtained in 0.1% TFA. Similar results have been obtained for other peptides and proteins (data not shown). • The mass spectrum for apoMyoglobin (32 pmol) in basic solution shows a shift to higher m/z charge states, relative to low pH. This effect varies with the peptide and protein, and is dependent on conformation (Winston and Fitzgerald, 1998; Konermann and Douglas, 1997).

  5. Comparison of TFA, NH4OH, and NH4 HCO3Eluents for Peptide RP-HPLC Separations Conditions: 8µL (200ng each peptide); Zorbax Extend C18, 2.1 x 150 mm; 0.25mL/min; 5-60% B in 20 min; 25°C TFA Conditions: A- 0.1% TFA in waterB- 0.085% TFA in 80% AcN LLL-NH2 LLVY LLVF LLF pH~1.8 LHL LLG LRL LLL NH4OH Conditions: A- 20 mM NH4OH in waterB- 20 mM NH4OH in 80% AcN LLVY pH~10.5 LRL LLVF LLF LLL-NH2 LHL Absorbance (210 nm) LLL LLG NH4HCO3 Conditions: A- 20 mM NH4OH in waterB- 20 mM NH4OH in 80% AcN pH~8.1 LLVY/LLL LLF LLVF LHL LRL LLG LLL-NH2 • Separations of this peptide mixture at high or low pH showed comparable band widths and peak shapes, although retention was less for all peptides using NH4OH, compared to TFA. • Poor peaks shapes were observed for ammonium bicarbonate buffer. No improvement was observed using higher or lower concentrations. • Retention order and selectivities shifted markedly with changes in pH.

  6. Comparison of TFA and NH4OH For Peptide RP-HPLC \ ESI-MS Analysis Conditions: 4µL (50ng each peptide); Zorbax Extend C18, 2.1 x 150 mm; 0.25mL/min; 25°C 5-60% B in 20 min; Pos. Ion ESI- Vf 70V, Vcap 4.5 kV, N2- 35 psi, 12L/min, 300°C TFA Conditions: A- 0.1% TFA in waterB- 0.085% TFA in 80% AcN 0.1% TFA LLL-NH2 LLL LLG LLVF LHL TIC (150-1500 m/z) NH4OH Conditions: A- 20 mM NH4OH in waterB- 20 mM NH4OH in 80% AcN 20mM NH4OH LLL LLL-NH2 LLG LLVF LHL • TIC signals were enhanced many-fold using NH4OH, relative to using TFA in the mobile phase. • Use of 0.05% TFA did not significantly improve signal intensity. • Use of 5 and 50 or 100 mM NH4OH in the eluents did not significantly change the TIC signal, relative to 20 mM NH4OH. • Separations of this peptide mixture showed comparable band widths and peak shapes, using either high or low pH mobile phases. • The separation selectivity is significantly changed with a change in pH.

  7. Peptide RP-HPLC/ESI-MS Using NH4OH Yields Both Positive and Negative Ions Conditions: 4µL (50ng each peptide); Zorbax Extend C18, 2.1 x 150 mm; 0.25mL/min; 25°C; 5-60% B in 20 min; Pos. Ion ESI- Vf 70V, Vcap 4.5 kV, N2- 35 psi, 12L/min, 300°C Positive Ions LLL-NH2 LLVF LHL LLL TIC (150-1500 m/z) LLG Negative Ions LLL-NH2 LHL LLVF LLL LLG • Significant negative ion signal was observed using NH4OH as the mobile phase modifier. • No significant negative ion signal is observed using TFA in the mobile phase. • All of the peptides showed both positive and negative ion spectra, with each exhibiting the singly-charged molecular ion. • The intensity of the negative current was less than the positive ion by about 2-fold, with similar baseline noise.

  8. Comparison of Angiotensins RP-HPLC Separations with TFA, NH4OH, and NH4HCO3 Conditions: 4 µL sample (200 ng each); Zorbax Extend C18,2.1 x 150 mm; 0.25 mL/min; 25°C; 5-60% B in 20 min. Angiotensin I Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu Angiotensin II Asp-Arg-Val-Tyr-Ile-His-Pro-Phe Angiotensin III Arg-Val-Tyr-Ile-His-Pro-Phe TFA Conditions A- 0.1% TFA in water B- 0.085% TFA in 80%AcN AII+AIII AI Absorbance (210nm) NH4OH Conditions A- 20 mM NH4OH in water B- 20 mM NH4OH in 80%AcN AII AIII AI NH4HCO3 Conditions A- 20 mM NH4HCO3 in water B- 20 mM NH4HCO3 in 80%AcN AII AI AIII • Separations of this peptide mixture showed comparable band widths and peak shapes, using either high or low pH mobile phases, although retention was less for all peptides using NH4OH, compared to TFA. • The separation selectivity of the AII/AIII band pair was greater at high pH, due the greater retention shift by AII. Angiotensin II has an additional C-terminal aspartyl residue, compared to Angiotensin III. The acidic side chain of the aspartyl residue would be ionized at pH 10, enhancing the selectivity difference. • At intermediate pH, peak width is increased for AIII, relative to the other angiotensins.

  9. Comparison of Angiotensins RP-HPLC/ESI-MS With TFA and NH4OH Conditions: 2.5 µL sample (50 pmol each); Zorbax Extend C18,2.1 x 150 mm; 0.2 mL/min; 35°C; 15-50% B in 15 min.; ESI-MS: Vf 70V, Vcap 4.5 kV, N2- 35 psi, 12 L/min., 325°C Angiotensin I Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu Angiotensin II Asp-Arg-Val-Tyr-Ile-His-Pro-Phe Angiotensin III Arg-Val-Tyr-Ile-His-Pro-Phe Positive Ions TFA Conditions: A- 0.1% TFA in waterB- 0.085% TFA in 80% AcN AII+AIII AI TIC (150-1500 m/z) Positive Ions AIII NH4OH Conditions: A- 20 mM NH4OH in waterB- 20 mM NH4OH in 80% AcN AII AI Negative Ions NH4OH Conditions: A- 20 mM NH4OH in waterB- 20 mM NH4OH in 80% AcN • Positive ion TIC signals were enhanced about 8-fold using NH4OH, relative to using TFA in the mobile phase. • A smaller but clear negative ion signal was observed using NH4OH in the mobile phase. • No significant negative ion signal is observed using TFA.

  10. Comparison of Angiotensin I Mass Spectra:Effect of Solution pH and Ion Polarity • Positive ion mass spectra obtained using TFA or NH4OH appear very similar, with similar production of the +1, +2, and +3 angiotensin I molecular ions. • The angiotensin I negative ion spectra shows a comparatively weak -3 molecular ion, consistent with the structure of the peptide.

  11. Comparison of Aß Peptide RP-HPLC Separations at Low and High pH Conditions: 5 µL sample (100 pmol each); Prototype 300 Å Zorbax Extend C18,2.1 x 150 mm; 0.25 mL/min Amyloid ß Sequences: Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu17 Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly38 Val Val40 Ile Ala42 Thr43-COOH TFA Conditions, 25°C A- 0.1% TFA in water B- 0.085% TFA in 80%AcN 33-45%B in 30 min. Ab(1-38) Ab(1-40) Ab(1-42/3) Absorbance (210 nm) TFA Conditions, 80°C A- 10 mM NH4OH in water B- 10 mM NH4OH in 80%AcN 29-41%B in 30 min. Ab(1-43) Ab(1-40) Ab(1-42) Ab(1-38) NH4OH Conditions, 25°C A- 20 mM NH4OH in water B- 20 mM NH4OH in 80%AcN 26-38%B in 30 min. Ab(1-43) Ab(1-42) Ab(1-40) Ab(1-38) • At 25°C and low pH, the recovery of Aß(1-42) and (1-43) is very poor (5-10%). • Retention of all peptides were lower at high pH, for any given temperature. • Resolution of the Aß(1-42) and (1-43) peptides could be obtained at low pH/high temperature, or at high pH at lower temperature. • The retention orders of Aß(1-42) and (1-43) are reversed at low versus high pH.

  12. Ab(1-43) Ab(1-42) Ab(1-40) Ab(1-38) Ab(1-43) Ab(1-42) Ab(1-40) Ab(1-38) 1083.3 Max: 14562 4 100 1443.9 3 80 60 5 6 7 40 866.5 722.7 20 1461.3 1097.3 1193.8 0 500 1000 1500 m/z 1083.2 Max: 168346 722.5 866.8 4 100 5 6 619.5 7 80 1443.8 60 3 703.0 40 1088.7 871.2 726.2 20 875.5 1094.3 616.7 542.2 0 m/z 500 1000 1500 Comparison of Aß Peptide Separation With TFA and NH4OH: ESI-MS Signal Conditions: 2.5 µL sample (50 pmol); Zorbax Extend C18, 2.1 x 150 mm; 0.2 mL/min; 35°C; Pos. Ion ESI- Vf 70V, Vcap 4.5 kV, N2- 35 psi, 12 L/min., 325°C TFA Conditions, 80°C A- 0.1% TFA in water B- 0.085% TFA in 80%AcN 29-41%B in 30 min. TIC (300 - 2000 m/z) NH4OH Conditions, 25°C A- 20 mM NH4OH in water B- 20 mM NH4OH in 80%AcN 26-38%B in 30 min. Comparison of Aß(1-40) Mass Spectra With TFA and NH4OH TFA NH4OH • Signal intensity and signal-to-noise are much higher for high pH versus low pH conditions for the amyloid peptide separation. • The charge state of the amyloid peptides at the ESI interface are shifted to higher numbers (lowerm/z) using the high pH mobile phase.

  13. Effect of Mobile Phase and Ion Polarity on MS Signal Intensity 20 NH4OH Positive Ion NH4OH Negative Ion 15 10 Relative Response TFA Pos Ion = 1 5 0 LHL LGL LLL LLVF LLL-NH2 Ang I Aß(1-38) Aß(1-40) (No Neg ion available) Peptide • Summary of relative signal intensities for peptides that have been studied to date: • 2-20 X greater signal in NH4OH compared to TFA. • Increasing molecular mass of peptide yields greater improvement in NH4OH compared to TFA for positive ion TIC intensity. • Negative ion TIC in NH4OH has a comparable intensity to positive ion TIC in TFA. Conclusions • A novel HPLC column packing material, Zorbax Extend-C18, performed well for peptide separations using both high and low pH mobile phases. The high stability of the column permits the use of these aggressive mobile phases • Useful separations of peptides at high pH can be conducted using ammonium hydroxide as a mobile phase modifier. • Peptide retention is lower at high pH than at low pH, when comparing TFA and NH4OH as the mobile phase modifiers. Retention is intermediate at intermediate pH, using NH4HCO3. • The use of ammonium bicarbonate for intermediate pH separations is practical, but significant band broadening was observed. This may be due to silanol interactions, or other causes. • Peptide separations at high pH show significant selectivity differences compared to low pH. • Proteins and peptides yield high positive ion ESI-MS signal intensities in mobile phases modified with ammonium hydroxide and organic solvents. • RP-HPLC/ESI-MS of peptides, using ammonium hydroxide solutions, can give excellent separations, with high ESI-MS detection sensitivity. • ESI-MS detection using NH4OH generates high quality negative ion mass spectra. • The amyloid ß-peptides were well resolved and showed high recovery at high pH at 25°C. In comparison, low pH conditions require the use of high temperature for high recovery and effective separation of Aß(1-42) and Aß(1-43). • Acknowledgments: • Steve Krouse and Bud Permar lent excellent technical assistance. Dr. Anita Hong (AnaSpec) generously supplied some of the amyloid peptide samples. Drs. A. Apffel and W. Hancock (Agilent Labs) made several useful suggestions over the course of this work. References Kelly, M., Vestling, M., Fenselau, C., Smith, P. (1992) Org. Mass Spectrom. 27, 1143. Kirkland, J., (1998) Anal. Chem. 70, 4344. Konermann, L. and Douglas, D. (1997) Biochem. 36, 12296. Winston, R. and Fitzgerald, M. (1998) Mass Spectrom. Rev. 16, 165.

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