## 4 Discussion
### Analysis of Protein Groups, Peptide Sequences, and PSM Variations
For the CHO sample, PEAKS identified 1043 unique peptide sequences (35.8%) compared to 233 (8%) in Mascot, with 1638 sequences (56.2%) common to both. In the _E. coli_ sample, PEAKS identified 1435 unique peptide sequences (37%) versus 202 (5.2%) in Mascot, with 2238 sequences (57.8%) common to both. For albumin-depleted plasma, PEAKS identified 1101 unique peptides (50.5%) compared to 64 (2.9%) in Mascot, with 1017 sequences (46.6%) common. To ensure an accurate comparison of peptide sequences, a list of stripped peptides from all db.peptides.csv output files of PEAKS was generated. This procedure included removing N-terminal protein acetylation (+42.01), carbamidomethylylation of cysteine residues (+57.02), and methionine oxidation (+15.99). Higher protein sequence coverage was observed in PEAKS compared to Mascot, particularly for CHO and albumin-depleted plasma samples (Supplementary Materials Figures S5-S7). The results of our DDA-based study were evaluated by comparing them with relevant literature data (Table 1). In this study, the number of protein identifications from CHO cells was higher than in a previous study, despite our use of a shorter gradient and a stricter 1% FDR search [46]. While [PERSON] et al. [47] identified more proteins from _Escherichia coli_ compared to our findings, their study used longer chromatographic columns and ran samples with over three-fold longer gradients. In human plasma, [PERSON] et al. [48] reported more protein identifications (442 versus 187 in this study), which can be attributed to their use of more efficient depletion kits, longer columns, and extended gradient lengths during sample preparation and analysis. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{**N**} & \multirow{2}{*}{**Sample**} & \multirow{2}{*}{**LC**} & \multirow{2}{*}{**MS/MS**} & \multirow{2}{*}{**(Length/Particle Size)**} & \multirow{2}{*}{**Gradient Length**} & \multicolumn{2}{c}{**Analysis**} & \multirow{2}{*}{**Number of Proteins**} \\  & & & & & & & **Parameters** \\ \hline \multirow{2}{*}{1.} & \multirow{2}{*}{CHO cells} & Ultimate & \multirow{2}{*}{LTQ linear ion trap} & \multirow{2}{*}{C18 AQ (15 cm)} & \multirow{2}{*}{110 min} & Sequest, 5\% & \multirow{2}{*}{392 total} & \multirow{2}{*}{[46]} \\  & & & & & & & FDR & \\ \hline \multirow{2}{*}{2.} & \multirow{2}{*}{CHO cells} & Ultimate & \multirow{2}{*}{QTOF} & PepMap RSLC C18 & \multirow{2}{*}{48 or 90 min} & \multirow{2}{*}{ \begin{tabular}{} \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{} \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{} \end{tabular}  } \\  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\ \hline \multirow{2}{*}{3.} & \multirow{2}{*}{\ \begin{tabular}{} \end{tabular}  } & Ultimate & \multirow{2}{*}{LTQ-} & PepMap RSLC C18 & \multirow{2}{*}{280 min} & \multirow{2}{*}{ \begin{tabular}{} \end{tabular}  } & \multirow{2}{*}{800–1000} & \multirow{2}{*}{[47]} \\  & & & & & & & FDR & \\  & & & & & & FDR & & \\  & & &Increasing gradient lengths from 48 to 90 min enhanced separation and broadened peaks across all samples, consistent with previous findings [18; 26]. A clear dependence of the chromatogram on MS acquisition frequency was also observed. This relationship is supported by a study that compared the analysis of complex marine bacterial samples using classic and improved ion traps with double MS/MS acquisition. The authors reported that higher acquisition frequencies significantly increased the number of identified proteins in such complex samples [20]. A slight increase in protein identifications was observed in albumin-depleted samples compared to plasma samples without the removal of this abundant protein. Although the total ion chromatograms showed lower intensities (Figure 1b), the number of protein identifications was higher when using high-frequency MS acquisition modes (B and D) compared to the standard modes (A and C).