PCR Instrument-assisted Acidolysis for Monosaccharide Composition Analysis of Serum Glycans

This protocol describes the procedures where a PCR instrument-assisted acidolysis is used for releasing monosaccharides from serum glycans. The monosaccharide composition analysis is subsequently obtained by a HPLC method that separates and quanties all 1-phenyl-3-methyl-5-pyrazolone (PMP)-labeled monosaccharides in 10 μl serum in 20 minutes. The rapid heating, precise temperature control, and gradient heating properties of PCR instrument provides with consistent acidolysis and derivatization conditions for up to 96 samples simultaneously. The described workow takes approximately 4–5 h, up to 72 serum samples can be analyzed with one HPLC instrument per day.


Introduction
Four major types of human biomolecules include nucleic acids (including DNA and RNA), proteins, lipids and glycans. Unlike RNAs and proteins, glycan biosynthesis has no templates but depends on genes, nutrition, and other environmental factors in time and space [1]. As results, animal glycome is estimated to be 10 4 times larger than the proteome [2][3][4][5] and glycans are abundantly found in patients suffering cancerous and non-cancerous diseases. However, nearly all studies of serum glycans as possible disease biomarkers have been focused on resolving complicated glycan structures by complicated glycan preparation procedures plus expensive instrumentations, such as LC-MS [2,[5][6][7].
Despite there are many different types of glycans [8], all human glycans consist of up to 10 monosaccharides, i.e. sialic acid, N-acetyl galactosamine, N-acetyl glucosamine, galactose, mannose, fucose, glucose, xylose, glucuronic acid, and iduronic acid. However, few methods have been developed to quantify glycan contents or monosaccharide compositions in human sera or plasmas.
This protocol was originally developed from the corresponding author's laboratory at Washington University in St. Louis for glucosamine-and galactosamine-based, serum-or animal tissue-derived glycosaminoglycan (GAG) quanti cation purposes [9][10][11][12][13]. The assay was subsequently used for detecting contaminants in heparin [14,15]. We then discovered that signi cantly different quantity and compositions of glucosamine and galactosamine are present in the plasmas of human patients suffering lung, breast, and pancreatic cancers, respectively, [10,16]. Since the major glycans in human sera/plasmas are N-linked and O-linked glycans instead of GAGs, we subsequently developed a HPLC method that can quantify all other monosaccharides in addition to glucosamine and galactosamine released from serum/plasma glycans simultaneously [17][18][19][20].
Releasing monosaccharides from glycans is the bottleneck of monosaccharide composition analysis [21,22]. It is usually conducted in a sealed glass ampoule at 105-120 •C for 1-6 h [23] or in a PicoTag station [10]. We have developed a PCR instrument-assisted acidolysis method for hydrolyzing different types of plant and animal glycans previously [20]. This protocol describes the procedures where a PCR instrument-assisted acidolysis is used for releasing monosaccharides from serum glycans. The monosaccharide compositions are then obtained by PMP-labeling and HPLC analysis. PMP derivative reagent Weigh the correct amount of PMP powder and dissolve in methanol to form 0.5 M PMP derivative reagent. CRITICAL Prepare the PMP derivative reagent before use.
HCl (6 M) Prepare by careful dilution from concentrated HCl. It can be stored at 20-25 °C. CRITICAL When working with concentrated HCl, fume hoods must be used and proper protective measures need to be taken according to all relevant workplace regulations. NaOH (3 M) Prepare by careful dilution from 50% w/w NaOH solution. It can be stored at 20-25 °C.
CRITICAL When working with 50% w/w NaOH solution, fume hoods must be used and proper protective measures need to be taken according to all relevant workplace regulations.
HAc-NH 4 Ac buffer system Acetic acid (HAc)-NH 4 Ac buffer system is composed of 0.2 M NH 4 Ac and 1/20 v/v HAc. CRITICAL The HAc is essential in this buffer system, which will neutralize excessive NaOH after derivatization. It can be stored for one week at 4 ℃.
Monosaccharides standard stock solution (10 mg/mL) Weigh the correct amount of monosaccharides standard, dissolve in Milli-Q water, aliquot into tubes and store at -20 ℃.
Mobile phase A (100 mM NH 4 Ac) Add the correct amount of NH 4 Ac into 800 mL Milli-Q water, and adjust the pH to 5.5 with HAc.
Mobile phase B HPLC-grade acetonitrile.

Equipment
In nity Lab Poroshell 120 EC-C18 (100 mm length × 4.6 mm inner diameter, 2.7 μm particle size; Agilent) Milli-Q ultrapure water system (Millipore) PCR instrument (T100; Bio-Rad) Microcentrifuge (Pico17; Thermo Fischer Scienti c) Agilent 1260 Series high performance liquid chromatography system (Agilent) Vortex shaker (Vortex 2; IKA) Para lm PCR tubes EQUIPEMNT SETUP PCR instrument setup For serum glycan degradation, the temperature control is set to: 1. heating-up to 100 ℃ in 90 s; 2. hold on for 10 mins; 3. cooling to 4 ℃ in 120 s and hold on for 5 mins. For monosaccharides derivatization, the temperature control is set to: 1. heating-up to 70 ℃ in 90 s; 2. hold on for 40 mins; 3. cooling to 4 ℃ in 120 s and hold on for 5 mins.
HPLC system setup The LC system should be optimized in order to minimize dead volumes. Gradients should be optimized for the samples at hand, and the post sample column washing and re-equilibration should be adjusted to the system in use. As an example, below is a table of the speci c setup used in our laboratory on an Agilent 1260 Series HPLC. CRITICAL STEP In alkaline conditions, non-reactive PMP will exist in the form of salt in the system, which will reduce the e ciency of subsequent extraction, and lead to an increasing background for HPLC analysis. Anticipated Results