Automatic amide I frequency selection for rapid quantification of protein secondary structure from Fourier transform infrared spectra of proteins

Abstract

Here we report the development of a new neural network based approach for rapid quantification of protein secondary structure from Fourier transform infrared (FTIR) spectra of proteins. A technique for efficiently reducing the amount of spectral data by almost 90% is suggested to facilitate faster neural network analysis. Additionally, an automatic procedure is introduced for selecting only those regions within the amide I band of protein FTIR spectra, which can be best related to secondary structure contents by subsequent neural network analysis. Based on a given reference set of FTIR spectra from proteins with known secondary structure, a subset of merely 29 out of 101 amide I absorbance values could be identified, which lead to an improved prediction accuracy. The average prediction accuracy achieved for helix, sheet, turn, bend, and other is 4.96% which is better than that achieved by alternative methods that have been previously reported indicating the significant potential of this approach. Our suggested automatic amide I frequency selection procedure may be easily extended to identify promising regions from spectral data recorded by other spectroscopic techniques, like for example circular dichroism spectroscopy.

Matrix effects during monitoring of antibody and host cell proteins using attenuated total reflection spectroscopy

“…For quantifying protein levels in the low microgram scale per milliliter in cell culture against a HCP and media background, this approach has been successfully employed already 37. Likewise, the approach quantifying HCP against a mAb and media background should be achievable and was shown in previous experiments38; particularly since protein secondary structure can be compared using IR and antibody secondary structure exhibits exceptionally high content of beta‐sheet 44…”

“…Wavenumber range was then narrowed in 10 cm −1 intervals covering the Amide I band between 1,600 and 1,700 cm −1 . As antibodies are known to show a high beta‐sheet content, the wavenumber ranges indicative for elevated beta‐sheet content in proteins (1,620–1,635 cm −1 and 1,675–1,695 cm −1 ) were evaluated for mAb quantification 44. Furthermore, different optimization strategies using first and second derivative and visual inspection of peaks that correlated with mAb levels were used.…”

Insoluble Proteins

“…In addition to GlpF and MstX, both full-length MBP and E. coli thioredoxin (TxrA) can enhance production of membrane POIs [ 62 ]. If a fusion protein is undesirable for a given membrane POI, the following plasmids have been used successfully to produce membrane proteins in E. coli due to tight regulation of their promoters: pASK75, regulated by anhydrous tetracycline [ 43 ], and pRHA-67 (Xbrane Bioscience), tightly regulated by L -rhamnose [ 63 ].…”

“…haloplanktis TAC125 versatility has been improved by the development of genetically engineered strains with improved features as cell factories [ 59 , 60 ]. P. haloplanktis TAC125 was also the fi rst Antarctic bacterium in which an effi cient gene expression technology was set up, by the proper assembly of psychrophilic molecular signals [ 58 , 61 ] into a modifi ed E. coli cloning vector [ 62 ]. Several generations of cold-adapted expression vectors allow the production of recombinant proteins either by constitutive [ 61 ] or inducible profi les [ 63 ] and address the product toward any cell compartment or to the extracellular medium [ 64 ].…”

Fast Quantification of Recombinant Protein Inclusion Bodies within Intact Cells by FT-IR Spectroscopy

“…Specific regions in MIR spectra of proteins indicate secondary structures; in particular the amide I band (1700−1600 cm ‐1 ), belonging to the C‐O stretch vibration of the peptide linkage, is highly significant (9, 10) (Figure 1).…”