Quantification of protein glycation using vibrational spectroscopy.

Glycation is a protein modification prevalent in the progression of diseases such as Diabetes and Alzheimer's, as well as a byproduct of therapeutic protein expression, notably for monoclonal antibodies (mAbs). Quantification of glycated protein is thus advantageous in both assessing the advancement of disease diagnosis and for quality control of protein therapeutics. Vibrational spectroscopy has been highlighted as a technique that can easily be modified for rapid analysis of the glycation state of proteins, and requires minimal sample preparation. Glycated samples of lysozyme and albumin were synthesised by incubation with 0.5 M glucose for 30 days. Here we show that both FTIR-ATR and Raman spectroscopy are able to distinguish between glycated and non-glycated proteins. Principal component analysis (PCA) was used to show separation between control and glycated samples. Loadings plots found specific peaks that accounted for the variation - notably a peak at 1027 cm-1 for FTIR-ATR. In Raman spectroscopy, PCA emphasised peaks at 1040 cm-1 and 1121 cm-1. Therefore, both FTIR-ATR and Raman spectroscopy found changes in peak intensities and wavenumbers within the sugar C-O/C-C/C-N region (1200-800 cm-1). For quantification of the level of glycation of lysozyme, partial least squares regression (PLSR), with statistical validation, was employed to analyse Raman spectra from solution samples containing 0-100% glycated lysozyme, generating a robust model with R2 of 0.99. We therefore show the scope and potential of Raman spectroscopy as a high throughput quantification method for glycated proteins in solution that could be applied in disease diagnostics, as well as therapeutic protein quality control.

Using Collision Cross Section Distributions to Assess the Distribution of Collision Cross Section Values.

Careful transfer of ions into the gas-phase permits the measurement of protein structures, with ion mobility-mass spectrometry, which provides shape and stoichiometry information. Collision cross sections (CCS) can be obtained from measurements made of the ions mobility through a given gas, and such structural information once obtained should also permit interlaboratory comparisons. However, until recently, there was not a recommended standard form for the reporting of such measurements. In this study, we explore the use of collision cross section distributions to allow comparisons of IM-MS data for commonly analyzed proteins. We present measurements from seven proteins across three IM-MS configurations, namely, an Agilent 6560 IMQToF, a Waters Synapt G2 possessing a TWIMS cell and a modified Synapt G2 possessing an RF confining linear field drift cell. Mobility measurements were taken using He and N2 as the drift gases. To aid comparability across instruments and best assess the corresponding gas-phase conformational landscapes of the protein "standards", we present the data in the form of averaged CCS distributions. For experiments carried out in N2, CCS values for the most compact ion conformations have an interinstrument variability of ≤3%, and the total CCS distributions are generally similar across platforms. For experiments carried out in He, we observe the total CCS distributions to follow the same trend as observed in N2, while CCS for the most compact ion conformations sampled on the 6560 are systematically smaller by up to 10% than those observed on the G2. The calibration procedure (for TWIMS) yields TWCCS for native-like proteins which are largely similar to those obtained on DTIMS instruments. We collate previously reported values of CCS for these proteins in the form of histograms which bear a remarkable similarity to the CCS distributions, reflecting the conformational heterogeneity of proteins and also how conformer populations can be altered on transfer from solution to the detector. This gives concern for some caution when calibrating sample protein drift times simply with single numeric CCS values.

MhuD from Mycobacterium tuberculosis: Probing a Dual Role in Heme Storage and Degradation.

The Mycobacterium tuberculosis (Mtb) heme oxygenase MhuD liberates free iron by degrading heme to the linear tetrapyrrole mycobilin. The MhuD dimer binds up to two hemes within the active site of each monomer. Binding the first solvent-exposed heme allows heme degradation and releases free iron. Binding a second heme renders MhuD inactive, allowing heme storage. Native-mass spectrometry revealed little difference in binding affinity between solvent-exposed and solvent-protected hemes. Hence, diheme-MhuD is formed even when a large proportion of the MhuD population is in the apo form. Apomyoglobin heme transfer assays showed MhuD-diheme dissociation is far slower than monoheme dissociation at ∼0.12 min-1 and ∼0.25 s-1, respectively, indicating that MhuD has a strong affinity for diheme. MhuD has not evolved to preferentially occupy the monoheme form and, through formation of a diheme complex, it functions as part of a larger network to tightly regulate both heme and iron levels in Mtb.

The major secreted protein of the whipworm parasite tethers to matrix and inhibits interleukin-13 function.

Infection by soil transmitted parasitic helminths, such as Trichuris spp, are ubiquitous in humans and animals but the mechanisms determining persistence of chronic infections are poorly understood. Here we show that p43, the single most abundant protein in T. muris excretions/secretions, is non-immunogenic during infection and has an unusual sequence and structure containing subdomain homology to thrombospondin type 1 and interleukin (IL)-13 receptor (R) α2. Binding of p43 to IL-13, the key effector cytokine responsible for T. muris expulsion, inhibits IL-13 function both in vitro and in vivo. Tethering of p43 to matrix proteoglycans presents a bound source of p43 to facilitate interaction with IL-13, which may underpin chronic intestinal infection. Our results suggest that exploiting the biology of p43 may open up new approaches to modulating IL-13 function and control of Trichuris infections.

The Use of Mass Spectrometry to Examine IDPs: Unique Insights and Caveats.

A sizeable proportion of active protein sequences lack structural motifs making them irresolvable by NMR and crystallography. Such intrinsically disordered proteins (IDPs) or regions (IDRs) play a major role in biological mechanisms. They are often involved in cell regulation processes, and by extension can be the perpetrator or signifier of disease. In light of their importance and the shortcomings of conventional methods of biophysical analysis to identify them and to describe their conformational variance, IDPs and IDRs have been termed "the dark proteome." In this chapter we describe the use of ion mobility-mass spectrometry (IM-MS) coupled with electrospray ionization to analyze the conformational diversity of IDPs. Using the LEA protein COR15A as an exemplar system and contrasting it with the behavior of myoglobin, we outline the methods for analyzing an IDP using nanoelectrospray ionization coupled with IM-MS, covering sample preparation, purification; optimization of mass spectrometry conditions and tuning parameters; data collection and analysis. Following this, we detail the use of a "toy" model that provides a predictive framework for the study of all proteins with ESI-IM-MS.