Review N-glycoproteomics in plants: Perspectives and challenges Wei Song a, b , Maurice G.L. Henquet a , Remco A. Mentink b , Aalt Jan van Dijk a , Jan H.G. Cordewener a , Dirk Bosch a , Antoine H.P. America a , Alexander R. van der Krol b, ⁎ a Plant Research International, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands b Laboratory of Plant Physiology, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands ARTICLE INFO ABSTRACT Article history: Received 6 January 2011 Accepted 2 May 2011 Available online 12 May 2011 In eukaryotes, proteins that are secreted into the ER are mostly modified by N-glycans on consensus NxS/T sites. The N-linked glycan subsequently undergoes varying degrees of processing by enzymes which are spatially distributed over the ER and the Golgi apparatus. The post-ER N-glycan processing to complex glycans differs between animals and plants, with consequences for N-glycan and glycopeptide isolation and characterization of plant glycoproteins. Here we describe some recent developments in plant glycoproteomics and illustrate how general and plant specific technologies may be used to address different important biological questions. © 2011 Elsevier B.V. All rights reserved. Keywords: N-glycosylation Glycoproteomics N-glycans Glycopeptides Cell specific proteomics Mass spectrometry Contents 1. N-glycosylation of proteins imported into ER ....................................... 1464 2. Functions of N-glycans in the ER .............................................. 1465 3. Functions of N-glycans beyond the ER ........................................... 1465 4. Strategies in N-glycoprotein analysis: N-glycomics .................................... 1465 5. Strategies in N-glycoprotein analysis: N-glycoproteomics ................................ 1467 5.1. Gel based glycoprotein analysis ........................................... 1467 5.2. Complexity reduction by selective glyco-proteomics ............................... 1467 5.3. Lectin based selection of glycoproteins and glycopeptides ............................ 1468 5.4. Hydrazide based selection of glycoproteins and glycopeptides .......................... 1469 JOURNAL OF PROTEOMICS 74 (2011) 1463 – 1474 Abbreviations: ER, endoplasmatic reticulum; cgl, complex glycan less; GlcNAc, N-acetylglucosamine; OST, oligosaccharyltransferase; ERAD, ER-associated degradation; PNGase, peptide-N-glycosidase; UPR, unfolded protein response; MALDI-TOF MS, matrix assisted laser desorption/ionization time-of-flight mass spectrometry; PAGE, polyacrylamide gel electrophoresis; ConA, concanavalin A; WGA, wheat germ agglutinin; AIL, Artocarpus integrifolia lectin; PNA, peanut agglutinin; GalNAc, N-acetylgalactosamine; HRP, horseradish peroxidase; LC–MS/MS, liquid chromatography tandem mass spectrometry; CE, capillary electrophoresis; LTP, lipid transfer protein; RBCS, ribulose bisphosphate carboxylase; GO, gene ontology. ⁎ Corresponding author. Tel.: +31 317 482448. E-mail address: sander.vanderkrol@wur.nl (A.R. van der Krol). 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.05.007 available at www.sciencedirect.com www.elsevier.com/locate/jprot