Porcine pancreatin extract (PPE, EC 232-468-9) is widely used as a pancreatic enzyme replacement therapy (PERT) to compensate for deficiencies in conditions such as exocrine pancreatic insufficiency, chronic pancreatitis, and cystic fibrosis, where impaired absorption of dietary nutrients occurs due to inadequate secretion of digestive enzymes [1]. PERT includes multi-enzyme complexes such as porcine-derived PPE, which contains key digestive enzymes including porcine pancreatic lipase (PPL, EC 3.1.1.3) and porcine pancreatic α-amylase (PPA, EC 3.2.1.1) [2]. These enzymes play an essential role in digesting macronutrients, such as fats, starches, and proteins, thereby facilitating nutrient absorption and metabolism [3]. PPE has been shown to support digestive function, prevent indigestion, and help with weight management in patients with cystic fibrosis and chronic pancreatitis [4], [5]. Additionally, patients with exocrine pancreatic insufficiency associated with diabetes have also been reported to improve with the use of PPE [6]. PPL (molecular mass of 52 kDa), the primary enzyme in PPE responsible for fat digestion, is a triacylglycerol acyl-hydrolase that hydrolyzes triglycerides into di- and monoglycerides and free fatty acids prior to dietary fat absorption [3], [7]. A distinguishing feature of lipolytic enzymes, including PPL, is their enhanced activity in the presence of emulsified substrates [3]. Consequently, PPL-mediated hydrolysis involves three key steps: interfacial adsorption, interfacial activation, and catalysis [8]. Similarly, PPA (molecular mass of 55 kDa) in PPE facilitates the digestion of starch and other polysaccharides by hydrolyzing the α-(1,4)-glycosidic bonds in amylose and maltose molecules [3], [9]. PPA has been reported to interact with the N-glycans of glycoproteins, namely transferrin, fetuin, and ovalbumin, binding to complex, high-mannose (Man), and hybrid-type N-glycans [10]. The activity and stability of these enzymes are influenced by various factors, including N-glycosylation, which plays an important role in regulating enzyme function, solubility, and resistance to degradation [11].

Pancreatic enzymes undergo various post-translational modifications (PTMs), among which N-glycosylation is a key determinant of enzyme stability, activity, and degradation resistance [12], [13]. N-glycosylation commonly occurs in a specific consensus sequence characterized as Asn-X-Ser/Thr, where X can be any amino acid except Pro, which inhibits N-glycosylation [13]. N-glycans in mammalian glycoproteins are composed of N-acetylglucosamine (GlcNAc), Man, galactose (Gal), fucose (Fuc), and sialic acid. N-glycans can also be categorized according to N-glycan type (high-Man, hybrid, and complex-types) and the presence of diverse modifications (sialylation and fucosylation) [14].

Sialic acids, involved in the modification of N-glycans, are mainly found at the terminus of N-glycans in mammals. N-acetylneuraminic acid (Neu5Ac) is the predominant form of sialic acid at the termini of N-glycans in human therapeutic glycoproteins [15]. N-glycolylneuraminic acid (Neu5Gc), meanwhile, is not synthesized in the human body but can be metabolically incorporated from dietary sources, such as red meat [16]. Sialic acid affects the chemical, physical, and immunogenic properties of glycoproteins. Sialylation improves the thermal stability of glycoproteins, increases solubility through negative charge, and protects serum glycoproteins from degradation by capping terminal-Gal [17], [18]. Additionally, sialic acids possess highly specific recognition and binding properties for a variety of different cellular receptors involved in cell−cell, cell−matrix, and molecular recognition [19]. Fucosylation consists of core-fucosylation of GlcNAc linked to Asn in the N-glycan core and terminal-fucosylation of GlcNAc at the terminal position of the N-glycan [20]. Core- and terminal-fucosylation affect the flexibility of the antennary structure of N-glycans and mediate the regulation of cell growth, cell signaling, and protein−protein interactions [21], [22]. Core-fucosylation has also been implicated in immunity, malignancy, and biomarkers [23].

Although individual components of PPE, such as PPL and PPA, have been previously investigated for their biochemical properties, comprehensive N-glycosylation profiling of PPE as a multi-enzyme mixture remains limited. The N-glycans of pancreatin have been analyzed using DEAE (anion-exchange chromatography) and SP-Sephadex (cation-exchange chromatography) [24], two-dimensional (2D) nuclear magnetic resonance (NMR) [25], and gas chromatography (GC) [26]. However, detailed structural and quantitative analyses of its N-glycans have not been conducted with high sensitivity and accuracy.

In this study, N-glycans from PPE, PPL, and PPA were characterized using ultra-performance liquid chromatography (UPLC) and liquid chromatography (LC)–electrospray ionization (ESI)–higher energy collisional dissociation (HCD)–tandem mass spectrometry (MS/MS) with the use of a hydrophilic interaction liquid chromatography (HILIC) column. First, N-glycans were enzymatically released using peptide-N-glycosidase F (PNGase F), labeled with procainamide (ProA), and applied to UPLC for total profiling and absolute quantification. Then, LC-ESI-HCD-MS/MS was used to determine the structure of each N-glycan and obtain their relative quantities. In addition, to assess the functional role of N-glycans, we also performed enzyme activity assays before and after glycan removal.