Microwave Instrument-assisted Acid Hydrolysis plus HPAEC-PAD for Quantitative Glycan Monosaccharide Composition Analysis of Serum/Plasma Samples

This protocol describes the procedures where our published microwave instrument-assisted acid hydrolysis (MAAH) coupled HPAEC-PAD analysis are optimized for glycan monosaccharide composition analysis of serum/plasma samples. The optimized acid hydrolysis of serum/plasma samples takes only 10 min and 10 μl of acid and 2 μl serum/plasma samples. The monosaccharide composition analysis is subsequently accomplished by HPAEC-PAD analysis. Each step of the experimental procedures has been optimized with repeated tests of monosaccharide standards and serum samples. The described workow takes approximately 70-90 min, up to 48 serum/plasma samples can be analyzed with one HPAEC-PAD 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]. Glycans are abundantly present in blood circulations in patients suffering cancerous and non-cancerous diseases. Thus, glycans are rich source for biomarker discovery. 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].
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]. 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, [10,14]. 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 [15][16][17][18] for biomarker development.
Releasing monosaccharides from serum/plasma glycans is the bottleneck of monosaccharide composition analysis [19,20]. It is usually conducted in a sealed glass ampoule at 105-120 °C for 1-6 h [21] or in a PicoTag station [10]. To enhance the e ciency of the acid hydrolysis, we previously developed a MAAH coupled HPAEC-PAD analysis for monosaccharide composition analysis of 7 different types of polysaccharides [22]. In current study, we optimized the acid hydrolysis condition for releasing glycan monosaccharides from serum/plasma samples and the monosaccharide compositions are then obtained by HPAEC-PAD analysis.

Reagents
Deionized water (Millipore Mingche Q-Gard system, 18. The parameter setting for the power level of microwave is 100 watts, and the temperature is set at 100 °C, and the microwaving time is set for 10 min, respectively. HPAEC-PAD system With the ability to analyze samples at capillary, microbore, or standard ow rates (or any combination of two, in a dual system) at up to 5000 psi, HPAEC-PAD is the most adaptable ion exchange chromatography system. Thermo Scienti c™ Dionex™ ICS-5000+ system uses a 250mm chromatographic column consisting of 10 μm diameter nonporous beads to improve resolution. As an example, below is a table of the speci c setup used in our laboratory on an Thermo Scienti c™ Dionex™ ICS-5000+ chromatography system. CRITICAL STEP Make sure that all are added to the bottom without sticking to the wall and samples are mixed well, which is important for glycan acidolysis.
5. Use CEM Discover Hydrolysis microwave reactor to run acidolysis program, which takes 10 min.
CRITICAL STEP Make sure that nitrogen is lled with the reaction system to replace the air.
Sample transfer. TIMING 10 min for up to 10 samples 6. Transfer sample into 1.5 ml EP tube, after hydrolysis.
CRITICAL STEP Make sure that the sample is completely transferred out to avoid sample loss.
Acid removal. TIMING 30 min for up to 10 samples 7. Remove HCl by centrifugal evacuation.
CRITICAL STEP Make sure that HCl is removed to avoid affecting subsequent analysis.
? TROUBLESHOOTING. 11. Set up the HPAEC-PAD system to analyze the hydrolyzed sample as described above in EQUIPMENT SETUP.
CRITICAL STEP Make sure that the hydrolyzed monosaccharides can be analyzed within 3 days.

Troubleshooting
Troubleshooting advice can be found in following statement · Step 6 Problem: Sample loss; Solution: Add 20 μL DI water to transfer the remaining sample three times. · Step 7 Problem: Extra residues in hydrolyzed serum samples; Solution: Add 100 μL HPLC-grade methanol to remove the residual HCl for 3 times by centrifugal evacuation.
· Step 12 Problem 1: Early elution of monosaccharides; Solution: When NaOH is exposed to air too long, it absorbs CO 2 and the NaHCO 3 generated affects pH of eluting solution, which results in early elution of monosaccharides. NaOH solution should be prepared fresh.
· Step 12 Problem 2: No signal, weak signal or impurity peaks; Solution: Check the concentration of HCl. Serum cannot be fully hydrolyzed if the concentration of HCl is lower than 6 M. Over hydrolyzing samples by using higher than 6 M HCl could result in weak signal or impurity peaks.

Methodological investigation
We published a precise and convenient method for analyzing monosaccharide compositions of 7 different types of polysaccharides by MAAH plus HPAEC-PAD analyses previously [22]. Our ultimate goal is to use the microwave-assisted acid hydrolysis for serum/plasma monosaccharide composition analysis.

The effects of microwave power on monosaccharide standards
To test the effect of microwave power on monosaccharide standards, the established microwave conditions (100 W at 100°C for 10 min) without 3 mol/L HCl, are applied to 0.1 mg/ml mixed six monosaccharide standards. The t-test was performed on the A (no-microwave) and B (microwave) groups containing the 6 monosaccharide standards. We found that (Figure 1) there is no signi cant difference between the two group (p > 0.05), indicating that the microwave-power itself has no effect on both physical and chemical properties of each of the monosaccharides in the mixture at the condition of 100 W at 100°C for 10 min.
The effect of HCl concentrations on serum MAAH Since the glycans in the serum/plasma samples are present as thousand different kinds of glycoproteins and glycolipids, which are not pure polysaccahrides as those used in our previous publication [15], we tested the best HCl concentrations that should be used for microwave-assisted acid hydrolysis of serum/plasma samples.
We rst made a serum pool by combining 100 μL serum from each of 10 kidney cancer patients. Two μL serum x 6 from the serum pool are taken and 0, 1, 2, 3, 4 and 5 mol/L HCl ( nal concentration) are used for acid hydrolysis under the condition of 100 W at 100 °C for 10 min, respectively. The resulting supernatant at each HCl concentration is analyzed by HPAEC-PAD. The ion chromatogram is shown in Figure 2a. The experiment is repeated three times. The average peak areas of each monosaccharide obtained from the serum pool is calculated. We found that 4 mol/L HCl produces the highest yield of two basic monosaccharides (GalN and GlcN) but with less neutral monosaccharides (Fuc, Gal, Glc, and Man). In contrast, 2 mol/L HCl only partially releases the two basic monosaccharides (GalN and GlcN). We concluded that 3 mol/L HCl keeps a balance in releasing both basic and neutral monosaccharides ( Figure   2b).

The best volume of serum/plasma samples required for monosaccharide composition analysis
In searching for the best volume of serum samples for monosaccharide composition analysis, we made another serum pool by combining 100 μL serum from each of 10 uremia patients. In triplicate, 2, 5 or 10 μL sera from the serum pool are used for acid hydrolysis under the condition of 3 mol/L HCl, 100 W at 100 °C for 10 min, respectively. The resulting supernatant is analyzed by HPAEC-PAD. The ion chromatogram of serum monosaccharides of the serum pool is shown in Figure 3a. Under the same acid hydrolysis conditions, we found that the content of monosaccharides is increased with increased amount of sera used but the increase is not linear. We decided to use 2 mL serum for MAAH coupled HPAEC-PAD analyses for two reasons: 1. two mL serum is su cient for detecting all six monosaccharides; 2. For