In the early days of de novo sequencing, spectra were manually annotated using the b and y ion series in the acquired fragmentation spectra, where researchers visually inspected and labeled fragment ions to infer peptide sequences. This manual method was applied in the foundational metaproteomics study by Wilmes and Bond in 2004, where Q-ToF MS on excised 2D-gel spots identified porin, acetyl coenzyme A acetyltransferase, and a component of an ABC-type transport system, likely from dominant, uncultured Rhodocyclus-type organisms in activated sludge. Interestingly, modern user interfaces often retain support for this practice, as evidenced by the spectrum viewer in the CompOmics utilities library , as used in, amongst others, PeptideShaker , which supports metaproteomics data analysis .