10 May / June 2017 Results and Discussion Figure 2: Representation of calculated versus experimental glucose units for 92 N-linked glycans analysed. N-linked Glycan Release and Purification


A combination of ribonuclease B, ovalbumin, alpha-1-acid glycoprotein (human), alpha- 1-acid glycoprotein, fetuin (bovine), and asialofetuin (bovine) was dissolved in 50 mM AMBIC, pH = 7.6. The sample solution was heated at 100°C for five minutes to denature the proteins, then allowed to cool for five minutes. Trypsin digestion was performed with an overnight incubation at 37°C with an appropriate aliquot of enzyme solution added to the sample (1 mg TPCK treated trypsin per 100 µL AMBIC buffer, protein:trypsin = 20:1). Trypsin enzyme deactivation was performed by heating the sample at 100°C for five minutes. Enzymatic release of N-linked glycans was done with an overnight incubation at 37°C with an appropriate aliquot of PNGase F added to the sample (protein:PNGase F = 1 mg:6 IUB milliunits). Released N-linked glycans were separated from the rest of the solution by reverse-phase liquid chromatography using a C18 SPE column, then frozen and lyophilised.


Procainamide (ProA) Labelling of Released N-linked Glycans


A modified form of Klapoetke’s procedure [18] was used to label the released N-linked glycans with ProA. Labelling solutions were freshly prepared by adding 54 mg procainamide hydrochloride and 31.5 mg sodium cyanoborohydride per 500 µL of 7:3 (v/v) dimethyl sulphoxide and acetic acid mixture. ProA labelling was performed with an overnight incubation at 37°C with 50 µL aliquots of labelling solution added to each sample. Excess labelling solution was removed from the samples using MiniTrap G-10 size exclusion columns. The ProA


labelled released N-linked glycans were frozen and lyophilised, then stored until required for analysis.


HILIC Separation of ProA Labelled Released N-linked Glycans with SRM Detection of Chromatographically Resolved Glycoforms


Procainamide labelled N-linked glycan separation was achieved by means of a Nexera UFLC (Shimadzu) and a Halo Penta- HILIC column (2.1 mm id, 150 mm length, 2.7 µm particle size) (Advanced Materials Technology, Wilmington, DE, US). Column temperature was set to 60°C, and the solvent flow rate was 0.4 mL/min. Mobile phase A was a solution of 50 mM ammonium formate, 5% acetonitrile, and 0.1% formic acid in water and mobile phase B was acetonitrile. A linear gradient of 78%-48% mobile phase B in 75 minutes was used for all experiments. This gradient provided adequate separation for the wide variety of glycoforms in both simple and complex samples while expediting complete elution with minimal undesirable effects to either the experimental parameters or the resulting data. Samples were dissolved in 78% acetonitrile before injection in the UFLC system.


Selected reaction monitoring (SRM) and scheduled SRM experiments were conducted on a hybrid quadrupole/linear ion trap mass spectrometer (4000 QqQ, Applied Biosystems/MDS SCIEX, Foster City, CA, United States) using an electrospray source. N-linked glycan ion (Q1) values for each standard were calculated using GlycoWorkbench software, and a fragment ion (Q3) value of m/z 440.8 representing ProA and GlcNAc was used.


The standard glycoproteins used in this study were selected because they have well- characterised glycans. These standards also have a diverse array of glycoform structures, including high mannose, complex, and hybrid structures. Dextran ladder is also well known, commonly used as a reference standard, and was used to correlate time in minutes to glucose units. LC-MS analysis was performed on the released N-linked glycans from the combined glycoprotein standards, and 92 N-linked glycan signals were selected for analysis. The retention time in minutes for each glycan selected was converted into glucose units using the dextran ladder standard (Figure 1). After conversion of retention in minutes to glucose units was completed for each glycan, the amounts of the chromatographically influencing monosaccharides that comprise each glycan were determined, with separate values for applicable variations in linkage and position. Using the retention information in glucose units and the number of each monosaccharide species present in the glycans, a multi-variable linear regression analysis was performed. The objective of this form of analysis is to produce a model that represents the relationship between two or more independent explanatory variables (x), in this case the individual monosaccharide species, and a dependent response variable (y), in this case retention in glucose units, by fitting a linear equation to the experimental data. The model for multiple linear regression, given i observations, is written as:


yi = β0 + β1 x i,1 + β2 xi,2 + … + βp xi,p + ∈i


Using this linear regression model and one of several solving methods, a coefficient (β) for each x value can be calculated. (Table 1) The coefficient is the average change in the response variable for one unit of change in the predictor variable while all other model predictors are kept constant. Various relevant statistical values can also be calculated for each coefficient, including standard error, t-stat, P-value, and upper/ lower 95% confidence values, also shown in Table 1. The standard error is an estimation of the standard deviation of the coefficient, and is used as a measure of how precise the coefficient measurement is. All coefficients have a standard error below 0.2 and all but one have a standard error below 0.1, indicating the calculated coefficients show little variation across different cases. The t-stat is calculated by dividing the coefficient by the standard error. If the sample size exceeds 30 observations, as is the case with


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68