## 2 Materials and Methods
### Estimator Definitions  
All spectra were transferred to the Origin program, where they underwent treatment meaning the data for all the defined below estimators were gathered there. Thus, it allowed calculating estimators to concentrate on the secondary structure of the protein--the crystallinity estimator. All of them can be found in the literature and are defined as follows:  
* From ATR-FTIR spectra, the E\({}_{\text{C}}\)--intensity ratios within Amide I C=O stretching vibration of parallel \(\beta\)-sheet to antiparallel \(\beta\)-sheet A1620/A1699 [21]). Absorption values were calculated with a straight-line baseline from 1818 to 866 cm\({}^{-1}\).
* From pol-ATR-FTIR spectra, the E\({}_{\text{CPOL}}\)--intensity ratios for this were calculated from polarised spectra recorded at 0\({}^{\circ}\) and 90\({}^{\circ}\) for each absorption band [8]. Absorption values were calculated from Equation (1) with a straight-line baseline from 1818 to 866 cm\({}^{-1}\).
* From the XRD pattern, the E\({}_{\text{PXRD}}\)--is the sum of fitted peak areas of \(\beta\)-sheet domains divided by the sum of all fitted peaks [34]. Peak analysis was performed on spectra with a polygonal-line baseline from 10 to 35\({}^{\circ}\).  
It is essential to highlight that several methods of calculating crystallinity index and beta content can be found in the literature. Authors use three to six Gaussians to fit the XRD patterns [23; 35]. Before calculating E\({}_{\text{PXRD}}\), diffractograms (average ones calculated from three separate measurements) were resolved by fitting six Gaussian curves. No baseline subtraction was needed. These Gaussian curves were then attributed to silk I (two peaks centered at 19.22\({}^{\circ}\) (4.6 A) and 20.42\({}^{\circ}\) (4.3 A) for the MS sample), from silk II (three peaks at 24.82\({}^{\circ}\) (3.6 A), 27.98\({}^{\circ}\) (3.2 A) and 30.53\({}^{\circ}\) (2.9 A)) and amorphous domains (21.89\({}^{\circ}\) with average distances of 4.1 A). All six peaks were found in MS and artificially aged and historical samples. Fitting was performed with an r2 value of 0.999 (+/-0.001).  
Additionally, in the spectra collected by ATR-FTIR, the primary functional groups' estimators were monitored to give more insight into the provoked degradation pathways. We decided to use estimators proposed/defined and evaluated in our previous work [8; 33], which are:  
* Oxidation estimator: E\({}_{\text{AmideI}/\text{II}}\)--intensity ratios of Amide I C=O stretching vibration to Amide II N-H in-plane bending and C-N stretching vibrations A\({}_{1620}\)/A\({}_{1514}\) (baseline as above).
* Hydrolysis estimator: E\({}_{\text{COOH}}\)--band 1318 cm\({}^{-1}\) integral to band integral of CH3 bending vibration band located at 1442 cm\({}^{-1}\) P\({}_{1318}\)/P\({}_{1442}\) (baseline as above).  
Pol-ATR-FTIR allowed calculating dichroic ratio \(R\) (defined in Equation (2)) and order parameter \(S\) (defined in Equation (3))  
\[R=\text{A}\big{\|}/\text{A}\bot \tag{2}\]\[S=\frac{R-1}{R+2} \tag{3}\]  
The \(R\) parameter allows us to estimate the degree of dichroism; \(R\) = 1 means no dichroism, and \(R\) = 0 or to infinity, respectively, represents the maximum dichroism for completely parallel or perpendicular orientation of IR-activated transition moments to the molecule's axis. The \(S\) parameter investigates the degree of order in the sample's secondary structure sub-domains; it is set to 0 for the disordered structure. Depending on the same moments of transition geometry to the molecule's axis, it equals 1 when they are parallel and \(-\)0.5 when perpendicular [8; 36; 37].