Glycosciences Laboratory

Glycan glyco-probe

This tool has three parts: (A) Glyco-probe, (B) Tag moiety and (C) Glycan moiety.
Only the name of the glyco-probe is mandatory for basic data plotting (charts and tables), and the name must to be unique.
Other information such as glycan sequence information would be required in order to use the sequence-based filtering and sorting functions in Tabulation View.

To open Glycan Glyco-probe tool, click Tools in the main menu and select Glycan Glyco-probe.

Select Yes for entering a new glyco-probe information and Modify existing for updating the metada of pre-sotred glyco-probe, glycan moiety and tag data.

(A) Glyco-probe

Select from library - Users are able to create a new glyco-probe by copying the pre-saved data.
Glyco-probe ID – An entry ID of the glyco-probe.
Glyco-probe Name – Needs to be a unique name. (This item is compliant with MIRAGE compliant) Asterisk means ‘mandatory’ information.
Comment on purity – Any comment on the purity of the glyco-probe. (This item is compliant with MIRAGE compliant)
Other comment – Any other comment.
Backbone type – Users can select a category based on the glycan structure type (screenshot below). This list has been arbitrarily made in the Glycosciences Laboratory and is found useful in designating panels in histograms of binding data.
Enter other meta-data – Users are able to store other metadata of this glyco-probe. Please see Meta-data section.

(B) Tag moiety

Select from library – Users can select the pre-saved tag.
Or enter new Tag – A new entry by copying the pre-saved tag.
Tag Name – Needs to be a unique name.
Structure if known – Users are able to store any structure-related information.
Tag Nature – Select one from Natural / Synthesized / Unknown.
Comment – Any other comment.
Enter other meta-data button – Users are able to store other meta-data on this tag moiety. Please see Meta-data section.

(C) Glycan moiety

Enter another Glycan Moiety button – Users are able to enter multiple glycan structures per glyco-probe by clicking this button. The glycan structures are listed in the table.
Select from library – Users can select a pre-saved glycan.
Or enter new Glycan – A new entry by copying the pre-saved glycan.
Glycan Name – Needs to be a unique name
Sequence – A glycan sequence written in GlycoCT{condensed}, 2D TEXT, CFG-IUPAC, GlycoWorkbench Sequence (GWS) or WURCS format can be used for Filtering/Sorting in tabulation result view. (This item is compliant with MIRAGE compliant)
Or upload file – Users can write directly into the text box above, or upload file from here
Sequence format – Select the glycan sequence fromat from GlycoCT / 2D_TEXT / CFG-IUPAC / GWS / WURCS / Other
‘Composition and motifs (if known)’ button – Users can save composition and motif information if known.
GlyTouCan ID (if known) – GlyTouCan ID (This item is compliant with MIRAGE compliant)
Comment – Any other comment
Enter other meta-data button – Users are able to store other meta-data on this glycan moiety. Please see Meta-data section.

Meta-data

Users are able to enter meta-data, especially regarding the storage information, in (A) Glyco-probe, (B) Tag moiety and (C) Glycan moiety.

Glycan structure sequence format

GlycoCT{condensed}, 2D TEXT, CFG-IUPAC, Glyco Workbench Sequence (GWS) and WURCS formats can be used for entering glycan sequences.

*Please note: GlycanBuilder is implemented in CarbArrayART for drawing glycan structures. Users are able to convert the drawn structure into GlycoCT{condensed} and WURCS formats.

The table below shows Lacto-N-difucohexaose II (LNDFH-II) as an exemplar structure in six formats: Symbolic, 2D TEXT, GlycoCT{condensed}, Glyco Workbench Sequence (GWS), IUPAC condensed and WURCS formats.

**Lacto-N-difucohexaose II (LNDFH-II) - GlyTouCan ID: G70115XG**
Format	Structure
Symbolic
2D Text	*Please change "α" to "a", and "β" to "b" on CarbArrayART Glycan Probe Entry Tool.
CFG-IUPAC	Gal(b1-3)[Fuc(a1-4)]GlcNAc(b1-3)Gal(b1-4)[Fuc(a1-3)]Glc(?1-
GlycoCT{condensed}	RES 1b:x-dglc-HEX-1:5 2b:a-lgal-HEX-1:5\|6:d 3b:b-dgal-HEX-1:5 4b:b-dglc-HEX-1:5 5s:n-acetyl 6b:b-dgal-HEX-1:5 7b:a-lgal-HEX-1:5\|6:d LIN 1:1o(3+1)2d 2:1o(4+1)3d 3:3o(3+1)4d 4:4d(2+1)5n 5:4o(3+1)6d 6:4o(4+1)7d
GWS	freeEnd--??1D-Glc,p(--4b1D-Gal,p--3b1D-GlcNAc,p(--3b1D-Gal,p)--4a1L-Fuc,p)--3a1L-Fuc,p$MONO,perMe,Na,0,freeEnd
WURCS	WURCS=2.0/4,6,5/[a2122h-1x_1-5][a1221m-1a_1-5][a2112h-1b_1-5][a2122h-1b_1-5_2*NCC/3=O]/1-2-3-4-3-2/a3-b1_a4-c1_c3-d1_d3-e1_d4-f1

**6P-Man5 - GlyTouCan ID: G66870FO**
Format	Structure
Symbolic
2D Text	*Please change "α" to "a", and "β" to "b" on CarbArrayART Glycan Probe Entry Tool.
CFG-IUPAC	[6P]Man(a1-3)Man(a1-3)Man(a1-3)Man(a1-2)Man(?1-
GlycoCT{condensed}	RES 1b:x-dman-HEX-1:5 2b:a-dman-HEX-1:5 3b:a-dman-HEX-1:5 4b:a-dman-HEX-1:5 5b:a-dman-HEX-1:5 6s:phosphate LIN 1:1o(2+1)2d 2:2o(3+1)3d 3:3o(3+1)4d 4:4o(3+1)5d 5:5o(6+1)6n
GWS	freeEnd--??1D-Man,p--2a1D-Man,p--3a1D-Man,p--3a1D-Man,p--3a1D-Man,p--6?1P$MONO,perMe,Na,0,freeEnd
WURCS	WURCS=2.0/3,5,4/[a1122h-1x_1-5][a1122h-1a_1-5][a1122h-1a_1-5_6*OPO/3O/3=O]/1-2-2-2-3/a2-b1_b3-c1_c3-d1_d3-e1

Subarray layout

To open Subarray Layout tool, click Tools in the main menu and select Subarray Layout.

Select Yes for a new subarray layout entry. Alternatively Copy from existing to create a new entry by copying a pre-saved layout.

Entry page 1

Subarray Layout ID – CarbArrayART will generate a new ID automatically.
Layout name – A name of the subarray layout. This needs to be a unique name (e.g. “Subarray layout of Sialyl glycan Set 1”).
Comment – Any comment or description for this subarray layout.
Number of replicates – The number of replicate spots of each individual glyco-probe.
Number of levels (arrayed glyco-probe) – The number of concentrations or doses of arrayed glyco-probes.
Number of glyco-probes – The total number of glyco-probes printed in the array or subarray.
Columns and rows of spots – The number of columns and rows of spots in the array or subarray (e.g. “18” columns and “12” rows).

Entry page 2

Arrayed glyco-probe levels – A value of glyco-probe concentraion or doses (e.g. 100 uM or 2 fmol/spot).
Glyco-probe list – Pre-stored glyco-probe list. Users are able to search a specific glyco-probe by typing full or a part of the glyco-probe name or ID and clicking ‘Search’ button.
Subarray/Block layout – Where users define the printing location of each glyco-probe. The items listed in this table are: Row, Column, Glyco-probe, Level and Identification number of glyco-probes in this subarray.
- Row and Column – The numbers in Row correspond to the row position counting from the top, and the number in Column corresponds to the column position counting from the left of the subarray layout.
- Glyco-probe – The glyco-probes printed are placed by drag-and-drop from Glyco-probe list (left side table).
- Level – Users can select the level linked with Arrayed glyco-probe levels (green box).
- Identification number of glyco-probes in this subarray – The number indicates a group of glyco-probes. In tabulation result view, intensity values, which extracted from quantified scan data, are calculated based on the group of glyco-probes.

An example for sequentially printed subarrays:
Sequentially printed with 2 doses in duplicate

If a glyco-probe is printed sequencially at 2 doses twice (for each dose), the first 4 spots are grouped together as a glyco-probe A with the dose (concentraion) information.

In this case, users are only required to drag-and-drop glyco-probes in the printed location.

An example for non-sequentially printed subarrays:

<Non-sequentially printed with 2 concentraions of 6 replicates>

The image left indicates an example of a glyco-probes printed with 6 replicates in two concentrations: (dark blue circles) 100 uM labelled as Level 1 in the table below, and (light blue circle) 10 uM labelled as Level 2 in the table below.

In this case, users are required to:

Change the numbers of Identification number of glyco-probes in this subarray and Level respectively according to the array printing design.
Drag-and-drop the glyco-probe from Glyco-probe list to complete the block layout.

Array (Slide) layout

To open Slide Layout tool, click Tools in the main menu and select Array Layout.

Select Yes for a new array layout entry, and Copy from existing to create a new entry by copying the pre-saved layout.

Entry page 1

Name – A name of the subarray layout. This needs to be a unique name (e.g. ‘Array Layout of Sialyl glycan Sets 1&2’).
Comment – Any comment or description for this subarray layout.
Array geometry (the number of subarrays/blocks) – The number of rows and columns of subarrays or blocks.

Entry page 2

Users are able to customise the layout of the array by drag-and-drop a pre-entered subarray (below table) to the array layout (right figure).

GAL-based data entry for array geometry

Array geometry represents the layout of the spots on a microarray slide including the number of blocks (subarrays), numbers of columns and rows of spots in each array or subarray, and the list of glycan probes with their printed location in the block. This is similar to a GenePix Array List (often referred to as GAL file). Users can use a GAL file to enter the array geometry information into CarbArrayART by adding printing conditions for each glycan probe, such as the concentrations arrayed or dose arrayed per spot.

A template of GAL extended file is included in the software package, named ArrayGeometry_GALextended.xlsx.

To open GAL-extended File Entry tool, click Tools in the main menu and select GAL-extended File Entry.

Name – A name of the subarray layout. This needs to be a unique name (e.g. ‘Array Layout of Sialyl glycan Sets 1&2’).
Select the GAL-extended file (Excel file) by clicking Upload
Press OK once the file upload process is completed

Project

A project is used for storing the project and experiment related information. Users can categorise the data by using this project folder, for example store all the data related a grant project or collaborators.

A project can be created by Click ‘Create a new Project’ icon at the top page

Users are required to enter a unique project name here. Note that, a space is not allowed in a project name.

Please note: Users can rename a project anytime.

Collaborators, Task list, Events and Keywords information related to the project can be saved in the project property view.

Analyte (Sample metadata)

Users are able to record glycan binding sample metadata. An analyte can be created by clicking Create a new Analyte icon at the top page. Users are required to select Project where the Analyte are going to be saved.

Users are able to store sample related information.

New – Create a new sample metadata from scratch
Copy component – Copy from the saved sample metadata (parameter values are also copied)
Use Template – Create a new sample metadata using the template (parameter values are empty)

The image below is a screenshot of ‘Glycan microarray’ template.

Users can change the parameters (Descriptor and Descriptor group) by clicking 'Add Descriptor', 'Add Descriptor Group', 'Edit' and 'Delete' buttons.

Once Analyte metadata are filled, users are able to save the current version of the parameters (Descriptor Group and Descriptor) as a new template.

The saved template can be used when users create a new Analyte.

Experiment design

Users are able to record the experiment protocol using Experiment Design tool. An Experiment Design can be created by Click ‘Create a new Experiment Design’ icon at the top page.

Select the recorded sample (Analyte name) by clicking ‘Browse’ button to create a new experiment design.

New Design – Create a new Experiment Design from scratch
From Template – Create a new Experiment Design based on the template (parameter values are empty)
Copy existing design – Copy from the saved sample Experiment Design (parameter values are included)

There are four pre-stored templates. Prestored templates are : Fluorescence-labelled protein, Biotin-labelled protein, Sequential detection (unlabelled) and Tagged protein (His, Fc, Flag etc).

Pre-stored protocols are saved in Palette area including: Sample overlay, Fixation, Washing, Detection reagent, Drying and Scanning. Users can create an experiment protocol by selecting the items from Palette area and placing it to the Canvas area.

<Canvas Area>

Red Circle – Automatically generated. This indicates an Analyte (glycan binding sample)
Green Circle – Automatically generated. This indicates a Glycan Array Data

Users can edit the parameter values of each protocol (box) in the Parameter entry area.

Parameters and Parameter groups can be edited by clicking ‘Delete’, ’Add Parameter’ and ‘Add Parameter Group’, buttons.

Once Experiment Design is created, users are able to save the protocol and parameters as a new template.
The saved template can be used when users create Experiment Design.

Quantified array data

A ‘Glycan Array Data’ is saved under Analyte. When a user create a new Glycan Array Data, (1) GPR or Proscan data file(s) and (2) Array (slide) layout information are required.

Click the icon at the top main menu to create a new Glycan Array Data

Entry page 1: File type and other information

Name – A name of scan data such as experiment title (e.g. Human Adenovirus 52 SFK (100 ug/ml), Sialyl Glycan Array Sets 1&2, 01.05.19).
File Type – GenePix (gpr) or ProScan (Excel).
Number of Slides – The number of slides used per experiment (in many cases 1 slide per experiment).
Statistical Method – Select one from: Average or Elimination.
* Elimination method calculates an average value after removing the maximum and the minimum values per glyco-probe.
(For example) When glyco-probe is printed with 5 replicates, and the results values are:
Replicate A = 1000.0, Replicate B = 550.0, Replicate C = 1200.0, Replicate D = 870.0 and Replicate E = 2000.0. Then, Replicate B (minimum) and E (maximum) are excluded, and, the Elimination Value will be (1000.0 + 1200.0 + 870.0) / 3 = 1023.3
Signal to Use – Select one from: Median-B, Mean-B, Median or Mean (*’B’ stands for Background).

Entry page 2: Selecting array geometry

Array Layout – Select the pre-saved array layout used from the library (by clicking the “…” shown above in red box)

Entry page 3: Selecting analyte information

Entry page 4: Uploading scan file(s)

Upload the scan file(s) by clicking Upload files button.

GPR file (GenePix scanner)

Users are able to select the flourophore for GenPix files.

Excel file (Proscan)

Once the user clicks Finish, the uploaded files are processed and the overview page of tabulation results will be displayed.

File formats of binding intensities generated from scanner software

The list below shows the available file formats that can be accommodated currently in CarbArrayART for quantified intensity values.

GenePix Results 3
GenePix Export 3
GenePix Results 2
ArrayIt version 6.1.0
ProScanArray Express software

We will extend the file formats including ArrayVision software and other software in the future.

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