title: NMR profiling of biomolecules at natural abundance using 2D 1H-15N and 1H-13C multiplicity-separated (MS) HSQC spectra.
abstract: 2D NMR (1)H-X (X=(15)N or (13)C) HSQC spectra contain cross-peaks for all XHn moieties. Multiplicity-edited(1)H-(13)C HSQC pulse sequences generate opposite signs between peaks of CH(2) and CH/CH(3) at a cost of lower signal-to-noise due to the (13)C T(2) relaxation during an additional 1/(1)JCH period. Such CHn-editing experiments are useful in assignment of chemical shifts and have been successfully applied to small molecules and small proteins (e.g. ubiquitin) dissolved in deuterated solvents where, generally, peak overlap is minimal. By contrast, for larger biomolecules, peak overlap in 2D HSQC spectra is unavoidable and peaks with opposite phases cancel each other out in the edited spectra. However, there is an increasing need for using NMR to profile biomolecules at natural abundance dissolved in water (e.g., protein therapeutics) where NMR experiments beyond 2D are impractical. Therefore, the existing 2D multiplicity-edited HSQC methods must be improved to acquire data on nuclei other than (13)C (i.e.(15)N), to resolve more peaks, to reduce T(2) losses and to accommodate water suppression approaches. To meet these needs, a multiplicity-separated(1)H-X HSQC (MS-HSQC) experiment was developed and tested on 500 and 700 MHz NMR spectrometers equipped with room temperature probes using RNase A (14 kDa) and retroviral capsid (26 kDa) proteins dissolved in 95% H(2)O/5% D(2)O. In this pulse sequence, the 1/(1)JXH editing-period is incorporated in to the semi-constant time (semi-CT) X resonance chemical shift evolution period, which increases sensitivity, and importantly, the sum and the difference of the interleaved (1)J(XH)-active and the (1)J(XH)-inactive HSQC experiments yield two separate spectra for XH(2) and XH/XH(3). Furthermore we demonstrate improved water suppression using triple xyz-gradients instead of the more widely used z-gradient only water-suppression approach.
authors.affiliation: Division of Pharmaceutical Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, United States. Electronic address: kang.chen@fda.hhs.gov.
authors.forename: Kang
authors.initials: K
authors.lastname: Chen
authors.affiliation: Laboratory of Bacterial Polysaccharides, Division of Bacterial Products and Allergenic Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, United States.
authors.forename: Darón I
authors.initials: DI
authors.lastname: Freedberg
authors.affiliation: Division of Pharmaceutical Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, United States.
authors.forename: David A
authors.initials: DA
authors.lastname: Keire
mesh_terms: D000465:Algorithms; D002247:Carbon Isotopes; D003903:Deuterium; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D014867:Water; D003903:Deuterium; D012259:Ribonuclease, Pancreatic
keywords: MS-HSQC; Multiplicity editing; Natural abundance; Triple-axis gradient water suppression