advanced network modeling approaches, such as cho glyco net, have further elucidated how the spatial organization and enzymatic distribution within the golgi apparatus contribute to these cell line ‐ specific glyco sylation patterns, providing mechanistic insights into the relationship between genetic variation and glycan heterogeneity ( kotidis et al. 2023 ). maldi ‐ tof ms profiles of low m / z range permethylated n ‐ linked glycans derived from cho ‐ k1, cho ‐ s, cho ‐ k1mab, and cho ‐ smab cells highlight changes in the abundance of oligomannose n ‐ glycans ( m / z 1500 – 2500 ). all molecular ions represent the singly charged and sodiated form [ m + na ]. data were obtained from the 50 % acetonitrile ( mecn ) fraction from a c18 sep ‐ pak. structures shown with brackets have not had their antennal location unequivocally defined. some n ‐ glycan structures are not annotate d in these spectra in the interest of clarity — fully annotate d spectra for these samples are shown in figures s1 – s4. n ‐ glycan composition of cho ‐ k1, cho ‐ s, cho ‐ k1mab, and cho ‐ smab cell lines. note: the data are derived from the 50 % acetonitrile ( mecn ) fraction and represent the mean of three biological replicates. maldi ‐ tof ms profiles of high m / z range permethylated n ‐ linked glycans derived from cho ‐ k1, cho ‐ s, cho ‐ k1mab, and cho ‐ smab cells highlight changes in the abundance of complex n ‐ glycans ( m / z 2500 – 8000 ). all molecular ions represent the singly charged and sodiated form [ m + na ]. data were obtained from the 50 % acetonitrile ( mecn ) fraction from a c18 sep ‐ pak. structures shown with brackets have not had their antennal location unequivocally defined. some n ‐ glycan structures are not annotate d in these spectra in the interest of clarity — fully annotate d spectra for these samples are shown in figures s1 – s4. the y ‐ axis on the right ‐ hand side shows the relative intensity of the largest signal compared with the largest signal in the m / z 1500 – 2500 range from the same spectrum ( shown in figure 1 ). relative n ‐ glycan composition of cho ‐ k1, cho ‐ s, cho ‐ k1mab, and cho ‐ smab cell lines. this data are representative of the 50 % mecn fraction and the mean of three biological replicates. error bars represent ± 1 standard deviation ( sd ). the results of two ‐ tailed unpaired t ‐ tests are shown above the columns ( n = 3 / group, nonsignificant ( ns ) = p > 0. 05, * p < 0. 05, * * p < 0. 01, * * * p < 0. 001 ). the bottom left panel shows data comparing abundances of oligomannose n ‐ glycans containing 5 – 9 mannose residues ( m / z: man5 = 1579, man6 = 1783, man7 = 1988, man8 = 2192, man9 = 2396 ). data in the bottom right panel compare the abundances of complex, bi ‐ antennary, and core ‐ fucosylated n ‐ glycans containing 0 – 2 galactose residues ( m / z: g0f = 1836, g1f = 2040, g2f = 2244 ). the cho ‐ k1 and cho ‐ s cell lines show distinc t patterns of oligomannose n ‐ glycan abundances, which are reflected in the respective antibod y ‐ producing daughter cell lines ( see figure 1 ). in the maldi ‐ tof ms spectra of cho ‐ k1 and cho ‐ k1mab cells, we observe the following pattern of growing relative abundances: man5 < man7 < man6 < man8 < man9 ( when abbreviated as man x, the presence of a glcnac2 chitobiose core is implied ). on the other hand, a different pattern emerges in cho ‐ s and cho ‐ smab cells, where the ms spectra show the abundances of oligomannose n ‐ glycan signals as: man5 < man7 < man8 < man9 < man6. the lower abundance of man8 and man9 structures in cho ‐ k1 and cho ‐ k1mab cells relative to the cho ‐ s and cho ‐ smab cells could suggest increased activity or expression of er and golgi ‐ resident mannosidases in cho ‐ s cells. an increased residence time of glyco proteins in mannosidase ‐ containing er and golgi cisternae might also explain these results. a small difference in oligomannose n ‐ glycan patterns is also present in the two cho ‐ s cell lines, where a higher abundance of man5 and man7, and a lower abundance of man9 in the parental cell line suggest increased mannosidase processing relative to the cho ‐ smab cells. another key difference between cho ‐ k1 and cho ‐ s cells and the antibod y ‐ producing daughter cell lines is the complexity and abundance of larger structures at high mass ranges ( see figure 2 ). the largest n ‐ glycans are observed in the maldi ‐ tof ms profiles of cho ‐ k1 and cho ‐ k1mab cells, where n ‐ glycans with up to 12 lacnac moieties ( m / z 7820 ) are clearly resolved. this is a significantly higher mass range than what is resolved in ms spectra for cho ‐ s cells, which contain signals corresponding to n ‐ glycans with up to 9 lacnac moieties ( m / z 5388, see figure s2 for full annotation ). interestingly, the cho ‐ smab cells appear capable of generating larger structures at higher mass ranges than the parental cho ‐ s cells, where the largest n ‐ glycan signals resolved are m / z 7633 ( see figure s4 for full annotation ) and m / z 5485, respectively. while the mass range for cho ‐ smab cells is slightly smaller than in the cho ‐ k1 and cho ‐ k1mab cell glyco profiles, the largest n ‐ glycans contain up to 14 lacnac moieties ( m / z 7633 ). overall, an analysis of normalized intensities confirms that cho ‐ k1 and cho ‐ k1mab cells produce more complex n ‐ glycans ( 26 % – 30 % ) than cho ‐ s and cho ‐ smab cells ( 22 % – 23 % ) ( see figure 3 ), however the only difference found to be statistically significant was between cho ‐ k1 ( 30 % ) and cho ‐ s ( 22 % ) cells ( * p < 0. 05 ). the greater abundance of complex structures is also apparent from the relative intensity of the largest signal in the m / z 2500 – 8000 range compared to the m / z 1500 – 2500 range, which is ~ 3× higher in the cho ‐ k1 and cho ‐ k1mab cell lines ( 36 % and 30 % ) than in the cho ‐ s and cho ‐ smab cell lines ( 11 % and 10 % ) ( see blue y ‐ axis in figure 2 ). these results suggest that the increased mannosidase trimming in cho ‐ s and cho ‐ smab cells does not necessarily translate into increased downstream processing by glyco syltransferases. on the other hand, the larger mass range observed for cho ‐ smab cells relative to cho ‐ s cells is not reflected in the overall increase in the abundance of complex structures ( 23 % and 22 %, respectively ), which was slight and not statistically significant. interestingly, cho ‐ k1 cells appear to have a higher abundance of complex glycans than cho ‐ k1mab cells, whereas the inverse pattern was observed for cho ‐ s and cho ‐ smab cells. however, neither pairwise comparison was statistically significant. the higher abundance of complex n ‐ glycans in the glyco profiles of cho ‐ k1 and cho ‐ k1mab cells relative to cho ‐ s and cho ‐ smab cells is also reflected in a higher level of core ‐ fucosylated, galactosylated, and sialylated structures ( see figure 3 ). with regards to fucosylation, cho ‐ k1 cells have more core ‐ fucosylated n ‐ glycans than cho ‐ s cells ( 24 % vs. 17 %, * p < 0. 05 ). similarly, cho ‐ k1 cells have more galactosylated n ‐ glycans than cho ‐ s cells overall ( 29 % vs. 19 %, * p < 0. 05 ). the other pairwise comparisons for core ‐ fucosylation and galactosylation were not statistically significant. an analysis of the core ‐ fucosylated, biantennary agalactosylated, mono ‐ galactosylated, and di ‐ galactosylated n ‐ glycans ( g0f, g1f, and g2f, respectively ), reveals that all three structures are present in greater abundance in cho ‐ s cells compared to the other cell lines ( see figure 3 ). the smaller mass range and the lower abundance of larger complex and highly elongated n ‐ glycans observed in cho ‐ s cells are likely to explain the increased relative abundance of these smaller n ‐ glycans. similarly to the differences in core ‐ fucosylation and galactosylation, neuac sialylated structures are significantly more abundant in cho ‐ k1 cells ( 25 % ) than in cho ‐ s cells ( 10 % ) ( * * p < 0. 01 ).