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PROTEOMICS
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| An integrated LC-MS system for rapid and reliable analysis of proteins in chromatography fractions |
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March 2002
M. Mustén, S. Renlund, H. Wadensten, I. Salomonsson, and S. Lindqvist
Amersham Biosciences, Uppsala, Sweden
Ettanä LC-MS System is a new, integrated liquid chromatography-mass spectrometry system comprising Ettan ESI-ToF (Electrospray Ionization Time-of-Flight) mass spectrometer and either Ettan LC or Ettan microLC chromatography systems. Ettan LC-MS enables fast mass spectrometry with high sensitivity and high mass accuracy; these features ensure high-quality mass spectra, which enables reliable identification of proteins and peptides by peptide mass fingerprinting. Ettan LC-MS is also an ideal analysis tool in protein purification to determine the molecular weight and purity of chromatography fractions, eliminating the need for confirmational analysis by electrophoresis.
Introduction
Mass spectrometry is an excellent tool for protein identification and has contributed to speeding up the proteomics research phase in drug discovery. Electrospray ionization mass spectrometry (ESI MS) provides a faster and more accurate tool for analyzing liquid chromatography fractions during protein purification than traditionally used techniques, such as electrophoresis. The combination of a high-performance liquid chromatography system with an ESI mass spectrometer, possessing peptide mapping capability, is ideal for the analysis of post-translational modifications, drug candidates, micro-heterogeneities in recombinant proteins, and confirmation of the identity of proteins purified from natural sources.
Ettan LC-MS System (Fig 1) integrates a high-performance electrospray ionization time-of-flight mass spectrometer (Ettan ESI-ToF) with a microbore (Ettan LC) or capillary bore (Ettan microLC) chromatography system under the control of Ettan LC-MS software. Ettan LC and Ettan microLC are based on the ÄKTA™design chromatography platform. Both chromatography systems include inert biocompatible flow paths with minimum dead volumes to ensure high sample recovery.
Fig 1. Ettan LC-MS provides fast sample characterization and reliable identification of proteins and peptides in complex mixtures.
Ettan LC and Ettan microLC utilize new microbore SOURCE™ 5RPC columns for high resolution over a broad pH range, as well as µRPC C2/C18 and non-porous silica (NPS) columns that provide extremely fast, high-resolution liquid chromatography. Ettan microLC is also optimized for use with high-resolution capillary bore µRPC C2/C18 columns.
Other features common to both Ettan LC systems include optimized gradient formation that is accurate and reproducible at low flow rates. Ettan microLC includes a 30 nl capillary flow cell for the highest possible sensitivity and resolution. For maximum convenience, a conductivity monitor measures the true elution gradient in real time as it enters the column.
Chromatography fractions obtained from other ÄKTAdesign chromatography systems can be manually transferred to the Micro-Autosampler A-905 of Ettan microLC for automatic desalting prior to mass spectrometry analysis on Ettan ESI-ToF (Fig 2).
Fig 2. Configuration of Ettan LC-MS for desalting and concentration of up to 96 LC fractions prior to mass spectrometry. In this example, proteins are purified on an ÄKTAdesign chromatography system and fractions collected in a microplate. The microplate is transferred manually from Fraction Collector Frac-950 to the refrigerated Micro-Autosampler A-905 of Ettan microLC for automatic desalting and concentration before injection in Ettan ESI-ToF.
Ettan ESI-ToF is equipped with dual-ionization probes, which enable internal calibration and ensure high mass accuracy by providing the option of simultaneous spraying of sample and calibrants. The Ettan ESI-ToF mass spectrometer provides mass analysis over a wide mass range ensuring accurate molecular weight determination and unambiguous identification of proteins and peptides. Ettan ESI-ToF can be operated at a spectral rate of 100 spectra/s and is therefore suited for extremely fast LC-MS runs. A Trap Pulse ion guide function provides high sensitivity, which enables detection of low sample amounts and impurities.
Desalting using Ettan microLC
Mass spectrometry is an ideal tool for monitoring protein purification by determining the molecular weight and purity of chromatography fractions. However, the chromatography techniques normally used (e.g. ion exchange, size exclusion, or hydrophobic interaction) yield fractions at about neutral pH with relatively high salt concentrations. These buffer conditions are usually incompatible with ESI MS. Desalting of samples prior to ESI MS is therefore required to ensure high sensitivity and high quality. Automated sample desalting can be performed on Ettan microLC, which results in improved resolution of mass spectra generated from Ettan ESI-ToF.
Mass spectrometry used to monitor protein purification
Ettan LC-MS can provide high-resolution mass spectra of a wide range of high-molecular weight proteins (1). To demonstrate this capability, a protein mixture consisting of transferrin, RNAase A, a-chymotrypsin, cytochrome C, and lysozyme was separated by cation exchange chromatography on a Mini S™ PC 3.2/3 column connected to ÄKTApurifier 10 chromatography system. Sample fractions were collected in Fraction Collector Frac-950 and transferred to Ettan microLC for desalting on a MicroTrap™ column (Michrom Bioresources Inc., USA). Mass spectrometry was performed on Ettan ESI-ToF. Chromatography running conditions, the results of the protein separation, and the mass spectra are shown in Figure 3.
Fig 3. A. Purification of a protein mixture by cation exchange chromatography on Mini S PC 3.2/3. B. and C. Mass spectrometry of the cytochrome C and transferrin fractions showing crude spectra (lower panels) and deconvoluted spectra (upper panels).
The MicroTrap column, in combination with a special valve system and automatic injection of samples into Ettan ESI-ToF, proved to be a very efficient way of performing simultaneous desalting and on-line mass spectrometry. The cycle time was about 5 min. Consequently, fractions collected after ion exchange chromatography were analyzed in less than 30 min. Protein fractions proved to be very pure although sodium adducts were detected in the RNAse A-containing fraction (result not shown). Transferrin was present in two forms differing in mass by 284 Da, presumably representing two different glycosylation patterns.
Fast chromatography and mass spectrometry
Fast elution of peptides can be performed by using NPS RPC media. Figure 4 shows the mass spectrum of a tryptic digest of BSA after separation on an NPS C18 4.6 × 33 mm, 1.5 µm bead size column (Bischoff Chromatography, Germany). Although elution was achieved over 2.5 min, resolution was maintained and high-quality mass spectra were achieved. Moreover, Ettan ESI-ToF can be operated at a spectral acquisition rate up to 100 spectra/s for use with extremely fast LC/MS runs that generate spectral peak durations below 1 s.
Fig 4. A. Ion chromatogram (m/z 485–2000) of the total fragment spectrum of a tryptic digest of bovine serum albumin (BSA) analyzed on Ettan ESI-ToF. The tryptic digest was separated on a non-porous silica (NPS) RPC column prior to mass spectrometry. B. Spectra, from which the ion chromatogram was generated, showing doubly charged peptides.
Peptide mass fingerprinting and identification of post-translational modifications
Results on the rapid and precise peptide mapping of bovine β-casein using Ettan LC-MS have been reported (2). Bovine β-casein purified from porcine intestine was digested using modified trypsin. Peptide mass fingerprinting was performed on Ettan LC-MS using the NPS C18 column connected to Ettan ESI-ToF. The mass spectrometer was operated in positive mode at a spectral rate of 10 spectra/s and a capillary exit voltage of 150 V.
Bovine b-casein has five phosphorylated post-translational modifications. The peptide mass fingerprint, together with m/z values relevant to the structure of the peptides are shown in Figure 5. Protein database searches based on the peptide mass fingerprinting experiments revealed that 100% of the molecule was identified when compared with the theoretical tryptic cleavage of the protein. All fragments carrying phosphorylated post-translational modifications were reliably identified.
Fig 5.Ion chromatogram (m/z 100–2000) from the separation of the tryptic digest of bovine β-casein sample. The gradient was 0–60% buffer B (0.05% TFA in acetonitrile) over 2.5 min. The mass values (m/z) indicated for the peaks are related to the structure of the β-casein molecule.
Proprietary Ettan LC-MS software enables reliable protein identification
Protein databases allow rapid identification of proteins based on the spectra of the digested protein. Ettan LC-MS software not only controls Ettan ESI-ToF and Ettan LC systems, but also includes functions for protein identification by peptide mass fingerprinting. These functions are based on ProFound™ software (Proteometrics), which is optimized for use with Ettan LC-MS System. The protein database NCBI, which contains 500 000 non-redundant, as well as non-annotated entries, is included with Ettan LC-MS software. The database can be updated through Ettan LC-MS software via the Internet. With the excellent mass accuracy afforded by Ettan ESI-ToF, four or five peptides are usually sufficient for successful protein identification.
Conclusions
Ettan LC-MS offers a fast, excellent means of analyzing LC fractions in protein purification by determining molecular weight, and purity. Automated desalting of fractions prior to mass spectrometry can be efficiently performed using Ettan microLC. Ettan LC-MS allows very rapid peptide mapping and protein identification due to the high spectral rate and low peak durations provided by Ettan ESI-ToF. Identification of proteins and peptides in complex mixtures and fragments carrying post-translational modifications can also be achieved. Ettan LC-MS software not only controls Ettan ESI-ToF and an Ettan chromatography system, but also includes functions for protein identification by peptide mass fingerprinting.
References
1. Application Note: Mass spectrometry analysis of liquid chromatography fractions using Ettan LC-MS system, Amersham Biosciences, code number 18-1156-15, Edition AA (2001).
2. Application Note: Fast LC-MS using Ettan LC-MS for peptide mass fingerprinting and identification of post-translational modifications, Amersham Biosciences, code number 18-1156-16, Edition AA (2001).
| Ordering information | | |
| Ettan LC-MS System | 1 | |
| ÄKTApurifier 10 | 1 | 18-1400-00 |
| Micro-Autosampler A-905 | 1 | 18-5050-65 |
| MINI S PC 3.2/3 | 1 | 17-0687-01 |
| SOURCE 5RPC ST 2.1/150mm | 1 | 17-5208-10 |
| µRPC C2/C18 ST 1.0/150 mm | 1 | 17-6002-88 |
| µRPC C2/C18 ST 300 µm/150 mm (for use with Ettan microLC only) | 1 | 17-6002-89 |
| Bischoff NPS C18 1.5 µm, 4.6 × 14 mm | 1 | 17-6002-87 |
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