GUIDE TO LC MS - December 21 1 Spectrometry Analysis of Liquid Chromatography Fractions using Ettan LC MS System Henrik Wadensten, Inger Salomonsson, Staffan Lindqvist, Staffan Renlund, Amersham Biosciences, Uppsala, Sweden and Marketa Berkova, Analytica of Branford, Branford, Connecticut, USA. Summary spectrometry provides a faster and more accurate tool for analysing liquid chromatography fractions during protein purification than traditionally used techniques (e.g., electrophoresis). Liquid chromatography mass spectrometry (LC MS) using Ettan LC MS offers an excellent means of monitoring protein purification by determining molecular weight, identity and purity 4 3 2 1 5 4 3 2 1 25 2 15 1 5 No salt 25 5 75 1 125 15 175 NaCl 1 mmol/l 25 5 75 1 125 15 175 NaCl 1 mmol/l 25 5 75 1 125 15 175 Figure 1: Effect of, 1 and 1 mmol/l NaCl on the mass spectrum of myoglobin (5 µg/ml; 3 µmol/l). The figure demonstrates the importance of desalting liquid chromatography fractions to obtain high-quality mass spectra. of fractions. The procedure described involves initial protein purification on ÄKTA purifier 1, automated and rapid desalting of fractions on Ettan microlc, and MS analysis by electrospray ionization time-of-flight mass spectrometry (ESI-ToF MS) using Ettan ESI-ToF. Ettan LC MS provides high-quality mass spectra of a wide range of high molecular weight proteins including bovine serum albumin, cytochrome C, transferrin, ovalbumin, myoglobin, a monoclonal antibody and two -lactoglobulin variants. Introduction spectrometry offers an excellent means of monitoring protein purification and performing confirmation analysis of protein fractions collected from chromatographic purification. However, chromatography techniques normally used rely on the ion exchange, size exclusion or hydrophobic interaction properties of the proteins. With these techniques the mobile phase is ionic and at about neutral ph, (i.e., conditions that are poorly or not at all compatible with electrospray ionization mass spectrometry (ESI MS, Figure 1). We show results of an efficient technique for automated desalting in combination with mass analysis using Ettan ESI-ToF. The mass spectrometer is suited to mass analysis over a wide mass range. It is equipped with dual-ionization Ettan LC MS is a turnkey system combining ESI-ToF mass spectrometry with microbore and capillary chromatography. The system is easy to learn and operate and provides outstanding performance for obtaining accurate results in protein purification and proteomics. Circle 28
2 GUIDE TO LC MS - December 21 (a) Sample loading Equilibrate: 5% CH 3 CN with.1% TFA Load: Maximum 2 µg Wash 5% CH 3 CN with.1% TFA Pump P-95, incl. 2x SV-93 G1 A3 Filter P 1 2 3 4 6 5 B3 A1 A2 B1 B2 W1 INV-917(1) INV-917(2) MicroTrap column G2 (b) Sample elution (injection) Elute: 7% CH 3 CN with.5% acetic Pump P-95, incl. 2x SV-93 G1 A3 Mixer M-925 Buffers Filter P 1 2 3 4 6 5 Mixer M-925 B3 A1 A2 B1 B2 Buffers Cond ESI-ToF Figure 2: Configuration of Ettan microlc system showing flow path during (a) sample load and (b) elution (injection) operations. Microplates containing fractions collected at the ion exchange chromatography separation of the proteins were placed in A-95. After equilibration, 1 µl aliquots of each fraction were loaded onto the MicroTrap column according to (a) and subsequently eluted to the ESI-ToF (see (b)). UV MS electrospray W1 INV-917(1) INV-917(2) MicroTrap column G2 UV Cond ESI-ToF MS electrospray probes, enabling internal calibration by simultaneous spraying of sample and calibrants, which facilitates high mass accuracy. Ettan LC MS is a totally integrated system comprising Ettan ESI-ToF mass spectrometer and either Ettan LC or Ettan microlc. In this study, an LC MS system including Ettan microlc was used for automated sample desalting mass analysis. Products Used Amersham Biosciences products used in this study: Ettan LC MS system Enquire ÄKTApurifier 1 Enquire Mini Q PC 3.2/3 17-686-1 Mini S PC 3.2/3 17-687-1 Materials and Methods Protein purification: A protein mixture consisting of bovine serum albumin (BSA), cytochrome C, transferrin, ovalbumin and myoglobin was separated by cation exchange chromatography on a Mini S PC 3.2/3 column connected to ÄKTApurifier 1. A second protein mixture containing a monoclonal antibody and two -lactoglobulin variants was separated by anion exchange chromatography on a Mini Q PC 3.2/3 column. Sample concentrations ranged between.25 and.9 mg/ml (1 or 2 µl injected.) The elution buffer was 5 mmol/l Tris-HCl (in buffer B containing 1 mol/l NaCl), ph 7.5 for both column types. Flow-rate was.2 ml/min and the gradient was 1% B over 2 column volumes (CV) with a column wash at 1% B of 3 CV and a re-equilibration phase of 7 CV. The fraction size was.1 ml and samples were collected in microplates using Fraction Collector Frac-95. Desalting on Ettan microlc After purification on ÄKTApurifier 1, the microplates with collected samples were transferred to the autosampler of Ettan microlc for desalting and sample injection into the Ettan ESI-ToF mass spectrometer. Ettan microlc was configured with A-95, two switch valves (INV 917) and with a MicroTrap column connected to the second valve (Figure 2). Prior to each sample injection the column was equilibrated with a minimum of 15 µl of eluent A (5% acetonitrile containing.1% TFA). The equilibration continued during the first part of the autosampler injection. The flow-rate at sample application was generally 4 µl/min. Samples (<1 µl, <2 µg) were applied to the column and subsequently washed with.2 ml of eluent A to remove salt. At sample elution, the flow-rate was lowered to 1 µl/min and the concentration of eluent B (7% acetonitrile with.5% acetic acid) was raised in one step to 1% B. The conductivity monitor was positioned in the line going to waste for monitoring of the salt peak during the sample load and desalting phase of the operation (Figure 2). Spectrometry Analysis Ettan ESI-ToF was operated at pulse mode with a capillary exit voltage setting between 15 and 25 V depending on protein mass. Generally a larger protein demands the higher capillary exit voltage setting. Spectra were acquired at 1 spectrum/s with a detector voltage setting of 185 V. Results Desalting and mass spectrometry analysis: The MicroTrap column positioned in Ettan microlc, in combination with a valve system and automatic injection of samples proved to be a very
GUIDE TO LC MS - December 21 3 spectrometry offers an excellent means of monitoring protein purification and performing confirmation analysis of protein fractions collected from chromatographic purification. efficient way of performing simultaneous desalting and on-line mass spectrometry analysis. The cycle time was about 5 min. Consequently, fractions collected after ion exchange chromatography were analysed in less than 3 min. The results of the protein separation and the mass spectrometry analysis are shown in Figure 3. Each mass spectrometry analysis is presented with the crude spectrum in the lower panel and the deconvoluted spectrum in the upper panel. Protein fractions 1 8 6 4 2. Transferrin RNAse A -Chymotrypsin Lysozyme Cytochrome C 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48 5. 1. 15. 2. Transferrin 74 76 78 8 82 6+ 5 5+ 4 7818 7832 47+ 46+ 45+ 44+ 43+ 42+ 41+ 13 14 15 16 17 18 4+ 84 19 2 Cytochrome C 1236. RNAse A 13683.3 Na-adducts +22 Da 115 12 125 13 132 134 136 138 14 1 12 14 16 18 2 Lysozyme 1437.6 136 138 14 142 144 146 148 15 23 24 25 26 27 1+ 8+ 1+ 8+ 7+ 7 8 9 1 11 -Chymotrypsin 25658 28 12 14 16 18 2 14 16 18 2 22 24 Figure 3: Separation of a protein mixture by cation exchange chromatography on Mini S PC 3.2/3 and results from the mass spectrometry analysis of the fractions corresponding to the main peaks in the chromatogram. Crude spectra are shown in the lower panels while the upper panels show deconvoluted spectra.
4 GUIDE TO LC MS - December 21 Ettan LC MS is suitable for the analysis of high molecular weight connecting a MicroTrap column in Ettan microlc for automatic proved to be very pure although sodium adducts were detected in the RNAse A-containing fraction. Transferrin was present in two forms differing 284 Da in mass, presumably representing forms with different glycosylation patterns. Monoclonal Antibody The monoclonal antibody sample was previously analysed by SDS-PAGE. However, when the sample was separated by liquid chromatography on the Mini Q column, two peaks were detected. The two peaks corresponded to two different variants of the monoclonal antibody with slightly different masses 15 73 Da and 15 132 Da, respectively (Figure 4). These peaks might represent different glycosylation patterns of the antibody. -lactoglobulin The two variants of -lactoglobulin, A and B, were effectively separated on the Mini Q column. es from the two forms differed.6 Da from the theoretical values. Spectra taken Monoclonal antibody variant 1 1573 Monoclonal antibody variant 2 15132 14 145 15 155 16 14 145 15 155 16 76+ 75+ 74+ 73+ 72+ 71+ 7+ 6 68+ 67+ 66+ 65+ 64+ 63+ 62+ 61+ 6+ 5 5+ 4 72+ 71+ 7+ 6 68+ 67+ 66+ 65+ 64+ 63+ 62+ 61+ 6+ 5 5+ 4 2 22 24 26 28 3 22 24 26 28 3 4 35 3 25 Test mix consisting of: Monoclonal antibody:.54 mg/ml -lactoglobulin:.9 mg/ml Sample volume: 2 µl Protein purification on ÄKTApurifier Column: Mini Q PC 3.2/3 A-buffer: 5 mm Tris-HCl, ph 7.5 B-buffer: 5 mm Tris-HCl, ph 7.5 in 1 M NaCl Wash: Flow-rate: 2 µl/min Gradient: 1% B, 2 CV Wash: 1% B, 3 CV Fractions: 1 µl Re-equilibrate: 7 CV 2 15 1 2 3 4 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 3 31 32 33 34 35 36 37 38 39 4 41 42 43 44 45 46 5. 1. 15. 2. Figure 4: Separation of a mixture of a monoclonal antibody and -lactoglobulin by anion exchange chromatography on Mini Q PC 3.2/3. The results from mass spectrometry of the fractions corresponding to the two first peaks in the separation are shown above the chromatogram. Crude spectra are shown in the lower panels while the upper panels show deconvoluted spectra.
GUIDE TO LC MS - December 21 5 proteins. Automated desalting was efficiently performed by injection of samples and MS analysis. from the analysis of the valley fraction between the peaks revealed exactly the same values as were analysed for the separated entities (Figure 5). Conclusions Ettan LC MS is suitable for the analysis of high molecular weight proteins. Automated desalting was efficiently performed by connecting a MicroTrap column in Ettan microlc for automatic injection of samples and MS analysis. Cycle times of approximately 5 min are possible. spectra of high quality were achieved revealing two different forms of a monoclonal antibody. The two forms of -lactoglobulin were successfully separated and analysed. 18278.4 18278.4 18364.3 Variant B Variant B Variant A Variant A G8, A134 G8, A134 D8, V134 D8, V134 18364.3 176 18 184 188 178 182 186 175 18 185 19 A17 A16 A15 A14 A13 A12 A11 1+ 1+ 12 14 16 18 1 12 14 16 18 2 22 12 14 16 18 4 35 3 25 2 Test mix consisting of: Monoclonal antibody:.54 mg/ml -lactoglobulin:.9 mg/ml Sample volume: 2 µl Protein purification on ÄKTApurifier Column: Mini Q PC 3.2/3 A-buffer: 5 mm Tris-HCl, ph 7.5 B-buffer: 5 mm Tris-HCl, ph 7.5 in 1 M NaCl Wash: Flow-rate: 2 µl/min Gradient: 1% B, 2 CV Wash: 1% B, 3 CV Fractions: 1 µl Re-equilibrate: 7 CV %B 8. 6. 4. 2. 15 1 2 3 4 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 3 31 32 33 34 35 36 37 38 39 4 41 42 43 44 45 46 15. 1. 15. 2. Figure 5: Separation of a mixture of a monoclonal antibody and -lactoglobulin by anion exchange chromatography on Mini Q PC 3.2/3. The results from the mass spectrometry analysis of the fractions corresponding to the two last peaks in the separation are shown above the chromatogram. Crude spectra are shown in the lower panels while the upper panels show deconvoluted spectra. The experimental value of the molecular weight difference of -lactoglobulin A and B variants was 86.1 Da, which only differs.6 Da from the theoretical value 86.4 Da.