N-linked glycosite profiling and use of Skyline as a platform for characterization and relative quantification of glycans in differentiating xylem of Populus trichocarpa

Abstract

Our greater understanding of the importance of N-linked glycosylation in biological systems has spawned the field of glycomics and development of analytical tools to address the many challenges regarding our ability to characterize and quantify this complex and important modification as it relates to biological function. One of the unmet needs of the field remains a systematic method for characterization of glycans in new biological systems. This study presents a novel workflow for identification of glycans using Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT™) strategy developed in our lab. This consists of monoisotopic mass extraction followed by peak pair identification of tagged glycans from a theoretical library using an in-house program. Identification and relative quantification could then be performed using the freely available bioinformatics tool Skyline. These studies were performed in the biological context of studying the N-linked glycome of differentiating xylem of the poplar tree, a widely studied model woody plant, particularly with respect to understanding lignin biosynthesis during wood formation. Through our workflow we were able to identify 502 glycosylated proteins including 12 monolignol enzymes and 1 peroxidase (PO) through deamidation glycosite analysis. Finally, our novel semi-automated workflow allowed for rapid identification of 27 glycans by intact mass and by NAT/SIL peak pairing from a library containing 1573 potential glycans, eliminating the need for extensive manual analysis. Implementing Skyline for relative glycan quantification allowed for improved accuracy and precision of quantitative measurements over current processing tools which we attribute superior algorithms correction for baseline variation and MS1 peak filtering.

Reversed-phase separation methods for glycan analysis

“…The use of both labels makes quantification by MS possible. These labels can be used in combination with HILIC separation [116, 117], but reversed-phase separations are also performed [118, 119]. Unfortunately, no information was given on the separation efficiency of glycans with these labels.…”

Xylose Migration During Tandem Mass Spectrometry of N-Linked Glycans

“…Extraction of glycans from crude xylem protein (250 μg) [21] and HRP (50 μg) [23] was performed as previously described. For the glycan preparation, the “spike-in” protocol described by Hecht, McCord, and Muddiman was applied [23].…”

“…Xyloses are primarily incorporated into the glycomes of plants, and across this field, there has been a renewed interest to investigate the relationship between glycosylation and biological processes [16, 17] Our group has extensively studied Populus trichocarpa to model flux through the monolignol biosynthetic pathway, which is important for the pulp and paper and biofuels industries [18–20]. We recently showed that approximately 50% of the enzymes involved in the monolignol pathway are glycosylated with occupancy levels between 10–100%, and work is ongoing to better define the role of glycosylation in regards to how it modulates enzymatic activity [21]. …”

Modeling cross-regulatory influences on monolignol transcripts and proteins under single and combinatorial gene knockdowns in Populus trichocarpa

“…The proteins they 489 identified include pPAL1, pPAL3, pPAL4, pPAL5, pC3H3, p4CL3, pCAD2, pCAld5H2, 490 pCCoAOMT1, pCCoAOMT2, pCCR, and pHCT1. Like phosphorylation, glycosylation 491 can regulate protein localization, functional activity, ability to form multienzyme 492 complexes, and stability [16]. 493 Glycosylation could explain some of the behavior we observed in the protein 494 abundance data.…”

“…This 486 post-translational modification was found to impact the activity of the PtrAldOMT2 487 protein but not its abundance. Loziuk et al, identified 12 monolignol proteins that 488 contain motifs for potential glycosylation in P. trichocarpa [16]. The proteins they 489 identified include pPAL1, pPAL3, pPAL4, pPAL5, pC3H3, p4CL3, pCAD2, pCAld5H2, 490 pCCoAOMT1, pCCoAOMT2, pCCR, and pHCT1.…”