Introduction

• Preface
• Glucose Unit (GU)
• The make up of the 2-D/3-D map
• Analyses of N-glycan structures
• Parameterization of unit contribution
• Code Number
• Acknowledgements

• Preface
We briefly explain the 2-D/3-D mapping method which has been developed for the structural determination of asparagine-linked oligosaccharides (N-glycans) in glycoproteins.
In this method, N-glycans are released from the protein portion and the reducing ends are fluorecent labeled with 2-aminopyridine. These pyridylamino (PA)-glycans are separated by HPLC using three different columns sequentially; and from the elution positions on three HPLC columns, the structures of PA-glycans are simultaneously estimated.
The structure of a sample PA-glycan can be estimated by comparing its elution position on the map with the positions of the known reference N-glycans plotted on the 2-D map. The sample PA-glycan and one of the candidate reference PA-glycans are co-injected into two HPLC columns to confirm a single peak. Furthermore, the sample PA-glycan are digested with several glycosidases, then the changes of the elution positions are again compared with those of the reference N-glycans.
This method is useful not only as an analytical procedure for N-glycan structures, but also as a means of isolating large scale samples for NMR spectroscopy or MS spectrometry.
Approximately 1mg of glycoprotein is used for the method. PA-oligosaccharides (pico moles or femto moles) are used for one run of HPLC analysis.
The identified N-glycan structures are coded with by code numbers with alphaneumeric characters (Code Number, e.g. 110.4F, or 3A1-300.8, etc.)
The new Web application contains data on approximately 500 different PA-glycans: It includes the structures, HPLC elution positions expressed in glucose units (GU) on ODS and amide-silica columns, relative molecular mass, code numbers, sources of samples, and references.

• Glucose Unit ( GU )
GU expresses essentially the elution time of a sample from an HPLC column. The elution time expressed in real time or volume can vary depending on the individual column, its age, or the batches of buffers used. The introduction of the GU is meant to reduce such variations.

Fig .1 Measurements of Glucose Unit (GU) values

Let us explain how to express elution time as a GU, using Fig.1. Both the ODS and the amide columns are first calibrated respectively with a commercial PA-derivatized isomalto-oligosaccharide mixture. Numbers (4, 5, 6, etc.) indicate the degree of glucose polymerization. Sample PA-glycan is then applied to the column. Red peaks show the elution positions of the samples. The elution time of the sample compared with those of the glucose oligomers and the GU are estimated. The next step is plotting the GU values; 14.7 on the X-axis and 6.7 on the Y-axis are plotted on the 2-D map as coordinates (red dot) and compared with the coordinates of known reference N-glycans (blue dots) plotted on the 2-D map.
Since we first published 113 different N-glycan structures and their GU values on the 2-D map in 1988, in Analytical Biochemistry we have not had any need to revise them. However, if you use columns different from ours (Shim-pack CLC-ODS column and Amide-80 column), it may be difficult to obtain the same GU values.

• The make up of th 2-D/3-D map
The 2-D mapping method is completely included in the 3-D mapping method.
(1) The PA-glycan mixture is separated on a DEAE (diethylaminoethyl) column according to its sialic acid content into neutral, mono-sialyl, di-sialyl, etc. This is the first step in the 3-D mapping method.
(2) Next, using the two different types of HPLC columns (an ODS and an amide column), sample PA-glycans fractionated on the DEAE column are further purified and their structures are simultaneously estimated. The procedure is called the 2-D mapping method. The elution time on the ODS column is expressed as a GU to be plotted on the X-axis. The GU value on the ODS column depends on the fine structure of each oligosaccharide.
Even oligosaccharides of the same molecular size can be separated on the X-axis. Separation profiles are very sharp.
(3) Each sample PA-glycan separated on the ODS column is applied onto the amide column, and the elution time is expressed as a GU to be plotted on the Y-axis. The GU value on the amide column (Y-axis) depends roughly on the molecular mass of each oligosaccharide.
(4) The coordinates of the sample PA-glycans are plotted on the 2-D map.
(5) For each group of different sialylation, the above processes (2)-(4) are repeated.
Let us explain further using Fig. 2.
(a) and (b): When all the coordinates are plotted for each sialylation group, a
2-D map is created for each of these groups separated by the DEAE column.
(c) and (d): It is important that for each 2-D map the HPLC elution conditions used ( the composition of buffers and gradient changes ) to obtain the X-and Y-coordinates are completely identical, so the coordinates can be transposed from layer to layer. If we plot all the coordinates from all the layers on a single 2-D map, the resultant map is very confusing. To avoid such confusion, 3-D plotting consisting of layers of 2-D maps is introduced, as shown in (d).

Fig. 2 The make up of the 2-D/3-D map

• Analyses of N-glycan structures
N-glycan structure can be expressed by a tree-like form which spreads its branches on a trunk of a tri-mannosyl core structure. The approximately 500 structures so far documented can be expressed as a Glycotree Diagram that joins all 61 different units of sugar residue.

Fig. 3 Glycotree diagram with the UC values

The structure of an unknown N-glycan is estimated by comparing its position on the 3-D map with the positions of the PA- reference N-glycans plotted on the 2-D map.
(1) A few candidates whose coordinates coincide with those of the sample PA-glycan within allowable error (±5%) are chosen by a computer search.
(2) The sample PA-glycan and one of the candidate reference PA-glycans are co-injected into two different HPLC columns to confirm a single peak.
(3) The sample PA-glycan and the candidate reference PA-glycan are simultaneously digested with several glycosidases. Their elution positions are compared again. The comparison is continued until both PA-glycans yield the common trimannosyl core.

Fig. 4 Structural analysis using a glycosidase digestion

We will continue our explanation using Fig. 4.
It is sometimes necessary to go beyond a direct comparison of the GU. For example, the coordinates of an unknown sample (green cross) are placed among three known structures (red dots), and the molecular weight of the three references are all the same. In these cases, co-injection with a reference PA-oligosaccharide is the most reliable solution. Moreover, a digestion method using several glycosidases is very useful. Although the elution positions of the three candidates are very close, after b-N-acetylhexosaminidase digestion, the elution positions of the resultant three N-glycans disperse as illustrated in Fig. 4. Therefore, we can establish the originally unknown structure by a series of transformations.

• Parameterization of unit contribution
The basic assumption used in the paramerterization of unit contribution is that the elution position of a given PA-glycan is represented by the sum of the contribution of each component monosaccharide unit (unit contribution =UC). Calculation to obtain UC parameters was carried out by a linear multiple regeression analysis. All 61 different UC values obtained for ODS and amide columns are diagrammatically expressed in GU as shown in Fig. 3.
The purpose of the Parameterization of UC is as follows:
(1) From a given structure, the GU value of the N-glycan can be assumed.
(2) These calculated UC values are useful in predicting glycan structure from an observed GU on the 2-D map.
Let us explain the case (1) using Fig. 5.
Although code No. 110.4F (ODS 8.9, amide 7.0 ) exists on the 2-D map, the smaller structure lacking galactose residue (unit number 28) does not exist on the map. However, if we use the UC value of the galactose residue (ODS 1.3, amide 0.9) , the position of this new structure on the map can be estimated easily.

Fig. 5 Prediction of coordinates of an unidentified PA-glycan based on the UC values

• Code Number
The code number of the PA-N-glycans consists of a set of several elements with the following meaning. The number to the right of the hyphen (here 300.8) represents the neutral N-glycan code number.

Fig. 6. Code Number

• Acknowledgements
The 2-D/3-D mapping method has been developed since 1980. We are greatly indebted to all our collaborators for their generous support and for their encouragement. Especially we would like to express our sincere gratitude to the following persons:
Prof. Yoji Arata who provided the structural basis by NMR analyses for our proposed structures obtained by the 2-D/3-D mapping method. Prof. Yuan Chuan Lee who gave us advice on the application of a theory related to "UC" and who continues to offer useful comments on our work. Dr. Hiroaki Nakagawa and Dr. Noboru Tomiya who devotedly supported our research during its difficult beginnings. Dr. Yoshiki Yamaguchi who gave us helpful advice in the development of this Web application. Members of FCCA who for 5 years have given us the opportunity to express our data related to PA-glycans on their Website. This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology; and CREST of Japan Science and Technology Corporation.

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