Glycoconj J (2006) 23:355–369 DOI 10.1007/s10719-006-8492-3 Distinctive characteristics of MALDI-Q/TOF and TOF/TOF tandem mass spectrometry for sequencing of permethylated complex type N-glycans ∗ Shin-Yi Yu # · Sz-Wei Wu # · Kay-Hooi Khoo Received: 26 October 2005 / Revised: 2 January 2006 / Accepted: 5 January 2006 C  Springer Science + Business Media, LLC 2006 Abstract Concerted MALDI-MS profiling and CID MS/MS sequencing of permethylated glycans is one of the most ef- fective approaches for high throughput glycomics applica- tions. In essence, the identification of larger complex type N- glycans necessitates an unambiguous definition of any modi- fication on the trimannosyl core and the complement of non- reducing terminal sequences which constitute the respective antennary structures. Permethylation not only affords analy- ses of both neutral and sialylated glycans at comparable ease and sensitivity but also yields more sequence-informative fragmentation pattern. Facile glycosidic cleavages directed mostly at N-acetylglucosamine under low energy CID, as implemented on a quadrupole/time-of-flight (Q/TOF) instru- ment, often afford multiple losses of the attached antenna resulting in characteristic ions related to the number of an- tennary branches on the trimannosyl core. Non-reducing ter- minal epitopes can be easily deduced but information on the linkage specific substituent on the terminal units is often missing. The high energy CID MS/MS afforded by TOF/TOF instrument can fill in the gap by giving an array of addi- tional cross-ring and satellite ions. Glycosidic cleavages oc- curring specifically in concert with loss of 2-linked or 3- # Shin-Yi Yu and Sz-Wei Wu contributed equally to this work. ∗ Dedicated to the late Prof. Yasuo Inoue, without whom the body of work represented by this article would not have been initiated in Taiwan. S.-Y. Yu . S.-W. Wu . K.-H. Khoo () Institute of Biological Chemistry, Academia Sinica, Taiwan e-mail: kkhoo@gate.sinica.edu.tw Tel: 886-2-2785-5696 Fax: 886-2-2788-2043 K.-H. Khoo National Core Facilities for Proteomics Research, Academia Sinica, Taiwan linked substituents provide an effective way to identify the branch-specific antennary extension. These characteristics are shown here to be effective in deriving the sequences of additionally galactosylated, sialylated and fucosylated termi- nal N-acetyllactosamine units and their antennary location. Together, a highly reproducible fragmentation pattern can be formulated to simplify spectral assignment. This work also provides first real examples of sequencing multiply sialylated complex type N-glycans by high energy CID on a TOF/TOF instrument. Keywords Glycan sequencing . High energy CID MS/MS . MALDI TOF/TOF . Mass spectrometry Abbreviations CID collision-induced dissociation EI electron impact ESI electrospray ionization FAB fast atom bombardment MALDI matrix assisted laser desorption/ionization MS mass spectrometry MS/MS tandem mass spectrometry Q/TOF quadrupole/time-of-flight LacNAc N-aectyllactosamine OMe O-methyl substituent or methoxy Introduction High throughput and robust MALDI-based mass spectrom- etry (MS) profiling is often the preferred, principal “first screen” strategy in defining the complexity and character- istics of a glycome [1]. In comparison with ESI-based MS analysis, the singly charged nature of the afforded molec- ular ions gives an added advantage in spectral simplicity and thus the ease of interpretation of a highly heteroge- neous glycosylation profile. From a perspective of glycomic Springer