Protein Microarrays for High Throughput Hydrogen/Deuterium Exchange Monitored by FTIR Imaging.

Proteins form the fastest-growing therapeutic class. Due to their intrinsic instability, loss of native structure is common. Structure alteration must be carefully evaluated as structural changes may jeopardize the efficiency and safety of the protein-based drugs.

Determination of Secondary Structure of Proteins by Nanoinfrared Spectroscopy.

Nanoscale infrared spectroscopy (AFMIR) is becoming an important tool for the analysis of biological sample, in particular protein assemblies, at the nanoscale level. While the amide I band is usually used to determine the secondary structure of proteins in Fourier transform infrared spectroscopy, no tool has been developed so far for AFMIR. The paper introduces a method for the study of secondary structure of protein based on a protein library of 38 well-characterized proteins.

Protein Structural Denaturation Evaluated by MCR-ALS of Protein Microarray FTIR Spectra.

The loss of native structure is common in proteins. Among others, aggregation is one structural modification of particular importance as it is a major concern for the efficiency and safety of biotherapeutic proteins. Yet, recognizing the structural features associated with intermolecular bridging in a high-throughput manner remains a challenge.

FTIR Imaging of Protein Microarrays for High Throughput Secondary Structure Determination.

The paper introduces a new method designed for high-throughput protein structure determination. It is based on spotting proteins as microarrays at a density of ca. 2000-4000 samples per cm and recording Fourier transform infrared (FTIR) spectra by FTIR imaging.

Amino acid side chain contribution to protein FTIR spectra: impact on secondary structure evaluation.

Prediction of protein secondary structure from FTIR spectra usually relies on the absorbance in the amide I-amide II region of the spectrum. It assumes that the absorbance in this spectral region, i.e.

Evaluation of protein secondary structure from FTIR spectra improved after partial deuteration.

FTIR spectroscopy has become a major tool to determine protein secondary structure. One of the identified obstacle for reaching better predictions is the strong overlap of bands assigned to different secondary structures. Yet, while for instance disordered structures and α-helical structures absorb almost at the same wavenumber, the absorbance bands are differentially shifted upon deuteration, in part because exchange is much faster for disordered structures.

Searching for a Better Match between Protein Secondary Structure Definitions and Protein FTIR Spectra.

Obtaining protein secondary structure content from high-resolution structures requires definitions and thresholds for the various parameters involved, typically hydrogen bond energy or length/angle and backbone φ/ψ angles. Several definitions are currently used and can have a profound impact on secondary structure content. Fourier transform infrared (FTIR) spectroscopy has its own sensitivity to molecular geometry.

A convenient protein library for spectroscopic calibrations.

While several Raman, CD or FTIR spectral libraries are available for well-characterized proteins of known structure, proteins themselves are usually very difficult to acquire, preventing a convenient calibration of new instruments and new recording methods. The problem is particularly critical in the field of FTIR spectroscopy where numerous new methods are becoming available on the market. The present papers reports the construction of a protein library (cSP92) including commercially available products, that are well characterized experimentally for their purity and solubility in conditions compatible with the recording of FTIR spectra and whose high-resolution structure is available.

Infrared imaging of high density protein arrays.

We propose in this paper that protein microarrays could be analysed by infrared imaging in place of enzymatic or fluorescence labelling. This label-free method reports simultaneously a large series of data on the spotted sample (protein secondary structure, phosphorylation, glycosylation, presence of impurities, etc.).