## 3 Results and Discussion
### Chromatography and Mass Spectrometry of ISP Extracts
The extracts from meat, obtained after marinating, contained a relatively higher content of hydrophobic or long peptides than brine. The brine, after marinating at a temperature of 12 \({}^{\circ}\)C, contained larger amounts of compounds forming peaks within a shorter retention time segment, than other brines did. This may indicate a larger extent of hydrolysis of herring proteins at 12 \({}^{\circ}\)C than in lower temperatures. This agrees with previous results revealing that proteolysis progress causes an increase in the relative area of peaks within shorter retention time intervals [27; 28]. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Sample**} & \multicolumn{2}{c}{**Relative Area of Chromatographic Peaks (\%) 1**} \\ \cline{2-3}  & **Retention Time Interval** & **Retention Time Interval** \\  & **15–34.99 min** & **35–60 min** \\ \hline Meat, 2 \({}^{\circ}\)C & 52.32 & 47.68 \\ Meat, 7 \({}^{\circ}\)C & 60.24 & 39.76 \\ Meat, 12 \({}^{\circ}\)C & 51.41 & 48.59 \\ \hline Brine, 2 \({}^{\circ}\)C & 69.72 & 30.28 \\ Brine, 7 \({}^{\circ}\)C & 71.22 & 28.78 \\ Brine, 12 \({}^{\circ}\)C & 93.36 & 6.63 \\ \hline All meat extracts 2,3 & 54.66 \(\pm\) 4.86 4 & 45.34 \(\pm\) 4.86 4 4  \\ All brines 2,3 & 78.10 \(\pm\) 13.23 & 21.90 \(\pm\) 13.24  \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Relative areas of peaks in the chromatograms of brine and meat ISP extracts, measured using RP-HPLC. The MALDI-ToF mass spectra of brine and meat ISP extracts within the \(m/z\) (mass to charge ratio) range up to 10 kDa are presented in Figure 2. Entire MALDI-ToF mass spectra are presented in Figures S7-S12 in the Supplementary Materials. MALDI-ToF serves mainly as a tool for the qualitative analysis of peptides, proteins, and their mixtures [38; 39]. Quantitation of individual proteins or peptides, although possible, is difficult, and requires many repeats to obtain a significant correlation between the amount of compound and the peak intensity [38]. On the other hand, mass spectrometry measurements are not affected by hydrophobic or any other interactions (in contrast to chromatography) and provide a precise mass to charge ratio. The last value measured using MALDI-ToF mass spectrometry should also be interpreted carefully. Proteins form not only singly protonated ions (M + H)\({}^{+}\), but also may be doubly protonated or appear as aggregates containing two or three analyte molecules with one proton [39; 40; 41]. Figure 1: RP-HPLC chromatograms of (black line) meat ISP extracts, and (red line) brine ISP-extracts from marinades ripened at (**a**) 2 \({}^{\circ}\)C, (**b**) 7 \({}^{\circ}\)C, and (**c**) 12 \({}^{\circ}\)C.  
Taking the above into account, we decided to interpret the MALDI-ToF mass spectra in a similar way to chromatograms, i.e., to compare relative areas of peaks within particular \(m/z\) segments. For the purpose of comparison, a few samples were obtained and analyzed in the same way we could use relative peak areas within defined \(m/z\) intervals. We did not utilize \(m/z\) lower than 500 Da. The matrix (in this experiment, sinapinic acid) clusters predominantly possess masses below 500 Da, although they may also reveal higher  
Figure 2: MALDI-ToF mass spectra of (black line) meat ISP extracts and (red line) brine ISP extracts from marinades ripened at (**a**) 2 \({}^{\circ}\)C, (**b**) 7 \({}^{\circ}\)C, and (**c**) 12 \({}^{\circ}\)C.  
masses [42; 43]. Relative areas of peaks within various \(m/z\) segments are presented in Table 5.  
Some results of the MALDI-ToF MS confirmed our interpretation of the RP-HPLC chromatograms. The brine after marinating at 12 \({}^{\circ}\)C contained the most peptides with \(m/z\) below 4000 Da, and the least compounds with \(m/z\) above 4000 Da, characteristic of polypeptides and intact proteins. This result is consistent with the RP-HPLC, revealing a lower amount of material with long retention times, compared to other samples.