Abstract
Introduction
CE-MS has shown considerable potential in protein research and pharmaceutical development, however, technical challenges of hyphenation of CE with MS have limited its use. Here, we have developed a sensitive CE-MS setup for peptide and protein analysis, that employs a novel and low-cost sheathless CE-MS interface and a poly(ethyleneglycol)-coated capillary. Using the CE-MS setup, we have achieved fast and efficient separation and sensitive mass analysis of individual components of a complex sample of pharmaceutically relevant human proteins including insulin, tissue factor, α-synuclein and a tryptic digest of bovine serum albumin. We have furthermore compared the performance of the CE-MS technique to UPLC-MS and demonstrate significant advantages of the CE-MS setup in terms of analysis time, sample consumption and separation capacity.
Methods
The sheathless CE-MS interface is based on the generation of an electric contact for both CE and ESI voltage through a narrow fracture close to the end of the CE capillary. The interface was mounted on a Nano ESI source of a hybrid Q-TOF instrument. To eliminate unwanted surface adsorption and/or interactions between polypeptide analytes and the inner capillary wall (L= 65 cm, 50 µm I.D.), a PEG coated capillary was made using novel covalent and electrostatically adsorbed coating method. Background electrolyte solution containing 0.1% formic acid was used throughout unless specified otherwise. Samples were injected hydrodynamically (50 mbar, 10 s) and the applied separation voltage was +30 kV for 12 min. No pre-treatment of sample mixtures analyzed was needed.
Preliminary Data
The simple and low-cost interface design allowed the generation of a stable ESI spray capable of ionization at low nanoliter flow-rates (45-90 nL/min) for high sensitivity MS analysis. By analysis of a model peptide (Leucine Enkephalin), a limit of detection (LOD) of 0.045 pmol/µL (corresponding to 67 attomol in a sample volume of ~ 15 nL) was obtained. The merit of the CE-MS approach was demonstrated by analysis of bovine serum albumin (BSA) tryptic peptides. A well-resolved separation profile was achieved and comparable sequence coverage was obtained by the CE-MS method (70%) compared to a representative UPLC-MS method (74%). Moreover, very low sample consumption and shorter analysis time makes the CE-MS method more preferable. The CE-MS setup was subsequently used to analyse a mixture of proteins of differing size and complexity consisting of insulin, tissue factor, α-synuclein and BSA. Efficient separation with very narrow peak widths, comparable to those obtained for small peptides, and high quality protein ESI mass spectra could be achieved using only a small amount of sample (30 fmol). Our findings underscore a considerable potential of the current CE-MS setup for high-sensitivity analysis of samples containing complex mixtures of intact proteins. Furthermore, the method was successfully applied to analyze a sample containing only 30 fmol of an intact therapeutic mAb (Rituximab) suggesting significant real-world applicability in biopharmaceutical research. Finally, by employing a native CE buffer (ammonium acetate, pH 6), we show that the CE-MS interface facilitates gentle ESI of proteins, opening up for native MS applications in combination with ion mobility and other gas-phase techniques to probe the conformational properties of proteins analysed by CE-MS.
Novel Aspect
A novel, simple and low-cost sheathless CE-MS interface for fast and sensitive analysis of complex peptide/protein mixtures
CE-MS has shown considerable potential in protein research and pharmaceutical development, however, technical challenges of hyphenation of CE with MS have limited its use. Here, we have developed a sensitive CE-MS setup for peptide and protein analysis, that employs a novel and low-cost sheathless CE-MS interface and a poly(ethyleneglycol)-coated capillary. Using the CE-MS setup, we have achieved fast and efficient separation and sensitive mass analysis of individual components of a complex sample of pharmaceutically relevant human proteins including insulin, tissue factor, α-synuclein and a tryptic digest of bovine serum albumin. We have furthermore compared the performance of the CE-MS technique to UPLC-MS and demonstrate significant advantages of the CE-MS setup in terms of analysis time, sample consumption and separation capacity.
Methods
The sheathless CE-MS interface is based on the generation of an electric contact for both CE and ESI voltage through a narrow fracture close to the end of the CE capillary. The interface was mounted on a Nano ESI source of a hybrid Q-TOF instrument. To eliminate unwanted surface adsorption and/or interactions between polypeptide analytes and the inner capillary wall (L= 65 cm, 50 µm I.D.), a PEG coated capillary was made using novel covalent and electrostatically adsorbed coating method. Background electrolyte solution containing 0.1% formic acid was used throughout unless specified otherwise. Samples were injected hydrodynamically (50 mbar, 10 s) and the applied separation voltage was +30 kV for 12 min. No pre-treatment of sample mixtures analyzed was needed.
Preliminary Data
The simple and low-cost interface design allowed the generation of a stable ESI spray capable of ionization at low nanoliter flow-rates (45-90 nL/min) for high sensitivity MS analysis. By analysis of a model peptide (Leucine Enkephalin), a limit of detection (LOD) of 0.045 pmol/µL (corresponding to 67 attomol in a sample volume of ~ 15 nL) was obtained. The merit of the CE-MS approach was demonstrated by analysis of bovine serum albumin (BSA) tryptic peptides. A well-resolved separation profile was achieved and comparable sequence coverage was obtained by the CE-MS method (70%) compared to a representative UPLC-MS method (74%). Moreover, very low sample consumption and shorter analysis time makes the CE-MS method more preferable. The CE-MS setup was subsequently used to analyse a mixture of proteins of differing size and complexity consisting of insulin, tissue factor, α-synuclein and BSA. Efficient separation with very narrow peak widths, comparable to those obtained for small peptides, and high quality protein ESI mass spectra could be achieved using only a small amount of sample (30 fmol). Our findings underscore a considerable potential of the current CE-MS setup for high-sensitivity analysis of samples containing complex mixtures of intact proteins. Furthermore, the method was successfully applied to analyze a sample containing only 30 fmol of an intact therapeutic mAb (Rituximab) suggesting significant real-world applicability in biopharmaceutical research. Finally, by employing a native CE buffer (ammonium acetate, pH 6), we show that the CE-MS interface facilitates gentle ESI of proteins, opening up for native MS applications in combination with ion mobility and other gas-phase techniques to probe the conformational properties of proteins analysed by CE-MS.
Novel Aspect
A novel, simple and low-cost sheathless CE-MS interface for fast and sensitive analysis of complex peptide/protein mixtures
| Original language | English |
|---|---|
| Publication date | 8 Jun 2016 |
| Number of pages | 1 |
| Publication status | Published - 8 Jun 2016 |
| Event | 64th ASMS Conference: 64th ASMS Conference on Mass Spectrometry and Allied Topics - San Antonio, TX 78205, United States Duration: 5 Jun 2016 → 9 Jun 2016 Conference number: 64 http://www.asms.org/conferences/annual-conference/conference-program |
Conference
| Conference | 64th ASMS Conference |
|---|---|
| Number | 64 |
| Location | San Antonio, TX 78205 |
| Country/Territory | United States |
| Period | 05/06/2016 → 09/06/2016 |
| Internet address |