We report an evidence of unusual N-glycan biosynthesis by MII/MX double knockout mice identified by a systematic glycomic approach applying two-dimensional LC mapping database and matrix dependent selective fragmentation (MDSF) technique in MALDI-TOF/TOFMS. Quantitative profiling of all N-glycan structures including minor components from MII/MX nulls, MII nulls, MX nulls and wild type mice yielded totally 37 kinds of N-glycans, and 27 structures of them and their molar ratios could be determined by 2D-LC mapping technique. However, it was suggested that 10 other structures can not be identified by common 2D-LC mapping database and their expression levels were too low to make further structural determination using enzymatic digestion. Our special interest has been focused on the precise structural characterization of 6 unknown N-glycans synthesized by MII/MX knockout mice among 10 unclear structures because they might involve some unusual glycoforms as a key to insight into mammalian N-glycan biosynthesis. A systematic structural characterization by using MDSF revealed that 6 unknown structures are unusual pseudo-hybrid-type N-glycans and two of them were completely new glycoforms. This result clearly indicates that survival of living organisms in the double knockout mice requires other enzymes to mimic complex-type structures and afforded unusual oligosaccharides in the deficient biosynthetic pathways. However, MII/MX double knockout mice could synthesize only unusual pseudo-complex-type in addition to common high mannose-type and hybrid-type N-glycans due to the lack of both two indispensable alpha-mannosidases, MII and MX, for the synthesis of all complex-type N-glycans. The present study clearly demonstrated that use of versatile MDSF method in MALDI-TOF/TOFMS greatly accelerates precise structural identification of unknown/unusual N-glycans isolated from other glycosyltransferase knock outs. We believe that the present approach by combining 2D-LC mapping and MDSF-assisted MALDI-TOF/TOFMS becomes a general and versatile protocol for functional protein glycomics studies using knockout/transgenic mice.