Detection of glycosylation and iron-binding protein modifications using Raman spectroscopy

Abstract

In this study we demonstrate the use of Raman spectroscopy to determine protein modifications as a result of glycosylation and iron binding. Most proteins undergo some modifications after translation which can directly affect protein function. Identifying these modifications is particularly important in the production of biotherapeutic agents as they can affect stability, immunogenicity and pharmacokinetics. However, post-translational modifications can often be difficult to detect with regard to the subtle structural changes they induce in proteins. From their Raman spectra apo-and holo-forms of iron-binding proteins, transferrin and ferritin, could be readily distinguished and variations in spectral features as a result of structural changes could also be determined. In particular, differences in solvent exposure of aromatic amino acids residues could be identified between the open and closed forms of the iron-binding proteins. Protein modifications as a result of glycosylation can be even more difficult to identify. Through the application of the chemometric techniques of principal component analysis and partial least squares regression variations in Raman spectral features as a result of glycosylation induced structural modifications could be identified. These were then used to distinguish between glycosylated and non-glycosylated transferrin and to measure the relative concentrations of the glycoprotein within a mixture of the native non-glycosylated protein.

Metabolic fingerprinting for diagnosis of fibromyalgia and other rheumatologic disorders

“…The discriminating power plot ( Fig. 3C, black line) for FM versus RA was dominated by a band centered at 840 cm Ϫ1 attributed to tyrosine residues in proteins (38).…”

Genetically Controlled Lysosomal Entrapment of Superparamagnetic Ferritin for Multimodal and Multiscale Imaging and Actuation with Low Tissue Attenuation

“…Typical Raman bands for phenylalanine appear at 1002 cm −1 , which are regarded as protein markers . The Raman spectra were normalized to the peak at around 1450 cm −1 , since this band has been shown not to fluctuate in signal strength in proteins . The Raman spectrum of the magnetite reference was taken from the Spectral ID 3.03 (Thermo Fisher Scientific) database.…”

On/off resonance Raman optical activity of human serum transferrin

“…As a result, the Raman spectrum of the holoTf (top left spectrum in Figure ) reveals four resonance enhanced bands at 1,170 cm −1 , 1,284 cm −1 , 1,506 cm −1 , and 1,608 cm −1 , which can be assigned to phenolate vibrational modes . Remarkably, the Fe‐O vibrations, found at ∼425 cm −1 , is not enhanced by the resonance effect. The corresponding ROA (top right spectrum in Figure ) bands are all positive and opposite in sign to the electronic circular dichroism of the resonant electronic transition (see top right Figure ) as predicted by the single electronic state (SES) theory .…”

“…The (bio)pharmaceutical role of Tf is to bind to other therapeutics with short half lives and improve the pharmacokinetics of these drugs by extending their activity . Previous Raman spectroscopy studies by Ashton et al focussed on the determination of the glycosylated status and iron binding of transferrin in off resonance conditions. In this study, we extend this work, to the best of the author's knowledge, for the first time to a combined on/off RROA study of HuTf.…”