br Introduction Glycoproteins represent a staggeringly

Introduction Glycoproteins represent a staggeringly small fraction of proteins analyzed by high-resolution techniques despite high concentrations at the cell surface and critical roles in many human diseases. Though one-half or more of all human proteins are predicted to contain a carbohydrate chain linked through an asparagine residue (N-linked glycan; Apweiler, Hermjakob, & Sharon, 1999), only ~2% of all human protein structures in the Protein Data Bank show at least the first three residues of what is likely a much larger N-glycan (14% of these are the crystallizable fragment of immunoglobulin G (IgG Fc), as of May 2018). Many reasons account for the dramatic underrepresentation in the structural database and in structure/function studies. These factors include: challenges associated with preparing glycoproteins, difficulties crystallizing glycoproteins or resolving disordered glycans, and the requirement of specialized techniques to characterize glycoprotein composition. Solution NMR spectroscopy is particularly well suited to explore the structure and function of glycoproteins. Furthermore, recent advances in protein expression and glycoprotein analyses surmount these barriers. The goal of this chapter is to describe practical considerations to prepare human glycoproteins with appropriate glycans for analysis by multinuclear solution NMR spectroscopy. Mammals target many proteins to the secretory pathway with a ~20 amino PTP Inhibitor IV signal peptide at the N-terminus that localizes the translating ribosome to an endoplasmic reticulum (ER) transmembrane pore complex (Moremen, Tiemeyer, & Nairn, 2012). The nascent polypeptide chain is imported into the ER lumen during which time the oligosaccharyltransferase activity associated with the pore complex recognizes a three amino acid sequence of [Asn/any nonproline residue/serine or threonine] and transfers a large 14-residue carbohydrate chain from a dolichol diphosphate donor to the Asn sidechain (Fig. 1). Modified polypeptides then fold in the lumen of the ER where the N-glycan remodeling continues. Most glycoproteins then proceed to the Golgi where those designated for secretion are exported to the surface of the cell. During this transport, other types of modification may occur, including O-linked N-acetylgalactosamine (O-GalNAc) modifications to Ser or Thr residues in flexible regions (Fig. 1). N- and O-glycans are further modified during transport and the types and extent of remodeling are due in large part to the enzymes expressed in the Golgi. As a result, most glycoproteins exhibit significant compositional heterogeneity. Glycans are integral to protein function and harbor epitopes for specific receptors, provide stability, assist folding, impact oligomerization, and change the structure and function of proteins (reviewed extensively: Varki, 2017). Furthermore, blocking N-glycosylation is fatal to human cells in culture. Given the essential role glycans adopt in protein function, it is crucial to develop methods to simplify glycoprotein production and analysis. Furthermore, the appropriate investigation of disease-related factors often requires proteins in a form as similar to the native form as possible, including relevant modifications (Baum & Crocker, 2009). The development of multiple powerful expression and analytical tools provides new opportunities to probe glycoproteins using solution NMR spectroscopy. Here we describe the techniques utilized by our lab and the various considerations of targeted glycoprotein studies. Here we present strategies to prepare and analyze glycoproteins by solution NMR spectroscopy that utilize advances in glycoprotein expression using cultured human cells, strategies to produce homogenous glycoproteins with stable isotope labels, advances in mass spectrometry to analyze glycan composition, and specific NMR experiments to probe glycans. As such, the references cited note details of the individual techniques (with references to videos, where available).