Fig. 2: The results from GlycanDIA workflow.

A To analyze GlycanDIA data, the MS1-centric method needs firstly to calculate the precursor ion mass and monitor it from the MS1 level (885.82 m/z, z = 2, in this example). Subsequently, the product ion yielded from this glycan (e.g., 1258.45 m/z) was also monitored from the MS2 spectra that isolated the precursor ion. A series of product ions were extracted simultaneously to validate the glycan sequence. The MS2-centric strategy in the GlycanDIA data analysis strategy relied on the glycan signature ions from an MS2 spectrum. B The peak alignment between glycan precursor isotopic masses at the MS1 level (left) and a series of glycan product ions at the MS2 level (right). The blue, purple, and red colors correspond to 885.82, 886.33, and 886.83 m/z, respectively. The product ions are color-coded to facilitate visualization. C More than 270 different N-glycans were identified using GlycanDIA, while the conventional DDA-based method only identified 200 glycans. Blue color indicates DDA, and yellow indicates GlycanDIA. The error bar showed the standard deviation of identified glycans from three technical replicate (n = 3) of the N-glycans mixture released from RNase B and human sera using two methods. Data are presented as mean ± SD. D The glycan quantification in MS1 and MS2 levels was evaluated. E GlycanDIA covered all the glycans identified from DDA runs. F Three heavy isotope-labeled N-glycan standards were mixed with the N-glycan pool released from RNase B and human sera. The GlycanDIA provides more consistent glycan identification compared to the DDA-based method. G The GlycanDIA also generates less variations in quantitative results. Boxes show 25th–75th percentiles, center lines represent medians, whiskers indicate min–max range from ten technical replicates (n = 10) of standard heavy isotope-labeled N-glycans using two methods. H Extracted compound chromatogram (ECC) shows the elution profile of 15 HMO standards. The full name of HMO standards can be found in Supplementary Table 2. I The example GlycanDIA results of two HMO isomers, 3FL and 2’FL. The legend for monosaccharide units and annotations can be found in Supplementary Table 4. Source data are provided as a Source Data file.