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1. Library Generator

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Ben Anderson edited this page Jun 27, 2024 · 11 revisions

Overview

Library Generator allows you to create lipid spectral libraries with in silico fragmentation.

Library Generator has several key features:

  1. Libraries are generated are in .msp file format, and are compatible with other mass spectrometry software such as MS-DIAL.
  2. Create MSn fragmentation trees for your lipid classes.
  3. Define whether a fragment is needed to reach a certain level of identification confidence and writes this to the final .msp library file(see Fragmentation Rules tab)

Select Library Tab

Create new libraries here.

New libraries start from the default template files found under the Resources/LibraryTemplates directory of your LipiDex 2 download.

Backbones tab

Define backbones with a chemical formula here.

Backbones are the starting point for building your lipid class. They are typically a chunk of your lipid that is not fragmented during your MSn activation.

The most common backbones are Glycerol (C3H5), Sphingoid (C3H2) and Sterol (C27H45).

Notice that the glycerol backbone is defined without any oxygens. You can define it to include the oxygens (C3H5O3), however you will have to define your fatty acyls as having one less oxygen, and in the fragmentation rule you will have to ensure correct stoichiometry so that the oxygen is lost as well. Overall, it is much more convenient to define the glycerol without any oxygens as C3H5.

You can define any backbone, and even create one with an empty Chemical Formula. In this case, it will be merely a connecting piece between your moieties and your headgroup.

Adducts tab

Adding an adduct

  • Adduct name

    • Must use the conventional adduct nomenclature, e.g. [M + H]+ or [M - 2H]2- or [M + Ac - H]1-
      • Must include capital M to indicate the parent, will fail otherwise.
      • Must have the M and any additional atoms or shorthand enclosed in square brackets, e.g. [M + H]
      • Must have a plus + or minus - to the right. Including the number 1 in 1+ or 1- is optional.
    • Shortened names (e.g. Ac for the acetate ion, CH3CO2) are accepted, but downstream processing in other modules must be aware of this.

  • Chemical formula

    • Must be a valid chemical formula.
    • IF you have a long formula like CH3CH2CH3 then it will be shortened to C3H8
    • Currently LipiDex 2 does not support using parentheses for multiplying the group within the parentheses by a number.
    • Don't use parentheses

  • Is Loss

    • Choose whether the adduct is an overall loss of mass from the parent.
    • For the electron-only adduct like [M]+ or [M]-, choose False

  • Polarity

    • Select positive or negative mode for this adduct. It must match the charge on the outside of the Adduct Name

  • Charge

    • This is the absolute value of the Charge. Even though the charge of [M-H]- is -1, just set Charge to 1

Glycan and peptide headgroups tab

Define glycan and peptide moieties to include in a lipid class.

Adding a glycan or peptide

  • Name

    • Can be the sequence or any other name

  • Type

    • select peptide or glycan

  • Sequence

    • Peptides can take any capitalized single-letter abbreviation for an amino acid
    • Valid glycan monomers are:
      • Hex - Hexose - C6H12O6
      • HexNAc - N-Acetylhexosamine - C8H15NO6
      • HexA - Hexuronic acid - C6H10O7
      • HexN - Hexosamine - C6H13NO5
      • Pent - Pentose - C5H10O5

  • Chemical formula modifier

    • Any additional gain or loss of a molecular formula
    • Losses can be specified by a - minus sign before the number
    • e.g. to lose a Water group, H-2O-1
    • Loss of some atoms and gain of other atoms can be included in one formula e.g. C2 O2 H-4 N-2

To add numerous peptides or glycans, it is worthwhile to access the Library's source file PolyHeadgroups.csv to directly add sequences.

Fatty acids tab

Define your fatty moieties

  • Any kind of 'fatty' moiety can be defined, including fatty acyl, ether, alkyls, or any custom group that behaves like a fatty moiety.
  • These classes will be iterated over through the full combinatorial space using a Combinatorial Fragmentation Rule, see later.

Adding a Fatty acid

  • Give it a name like 18:1 to indicate a typical fatty acyl.
    • It is recommended, though not required, to put relevant numbers of carbons, double bonds and oxidations in this Name.
    • The convention is [# carbons]:[# unsaturations];O[# oxidations], e.g. 18:2;O1
    • The Name DOES NOT impact the final mass of this moiety. Chemical Formula defines the mass.
  • Type
    • To construct a series of fatty moieties, name them all the same.
    • Default library includes Acyl, Plasmenyl, Plasmanyl, Sphingoid and Phytosphingoid Types
  • Enabled
    • Whether the entry will be included when generating libraries. Used to easily remove fatty moieties unlikely to exist in a lipid class.

Lipid Classes tab

This tab combines all the parts of a lipid that you defined in the Backbones, Adducts, Fatty Acids and Oligos tables into a final lipid class definition.

Adding a lipid class

  1. First give your lipid class an abbreviation and a full class name.
  2. Define the headgroup chemical formula, if applicable.
  • You can define your lipid class with no headgroup if you want, just leave it blank.
  1. Define the Adducts you want to have in your lipid class using the checkboxes to the right.
  2. Select one of your Backbones.
  3. Select the Optimal Polarity for your lipid class.
  • Select either Positive, Negative, Both or Neither.
  • This is the polarity mode of the instrument that gives the most structurally informative fragmentation.
  • LipiDex 2 uses this internally for identifications. The identification of a feature is derived from the Optimal Polarity if there is a choice between polarity modes, for example in polarity-switching experiments, or when the user acquires the same sample in separate modes and includes both.
  1. Select up to 4 Moieties to connect to the lipid.
  • Generally, these are fatty groups, like fatty acyls
  • However, you can select Peptides, Glycans or any of the other lipid classes as a moiety.
  • If you select another lipid class, then during in silico fragmentation it will undergo combinatorics at the top lipid class level and also at the sub lipid class level.

Fragmentation Rules Tab

After defining lipid classes and their adducts, the Fragmentation Rules tab will automatically generate all the Lipid class - Adduct combinations. These are the Top Levels.

Next, add a Spectrum to the Top Level by clicking Create new spectrum

  • Select the type of activation (HCD, CID, etc.)
  • Set the level of evidence for the spectrum
  • Select the type of Mass analyzer
  • If you are using polarity inversion methods, you can select the Polarity mode, but generally the Polarity should stay the same as the parent adduct.

Creating fragmentation rules

Select from 2 types of fragment rules:

1. Single component fragment rule

  • This rule creates one fragment entry based on the parts of the lipid you include or leave out with the checkboxes.
    • Headgroup
    • Backbone
    • Each of the 4 attached Moieties, depending on whether one is specified in the lipid class.
    • The Adduct
      • If you retain the Adduct, then it is defined as a Neutral Loss in the .msp file fragment comment.
      • If you don't include the Adduct, then it is a Fragment in the comment.

2. Combinatorial fragment rule

  • Used for either a Neutral Loss or Fragment of the attached Moiety type

    • For example, triacylglycerol class TG has 3 Fatty Acyl moieties. This rule will create a lipid of each combination of Fatty Acyls given in the Fatty Acid class.
    • Another example, Alkanyl-TG class contains a plasmanyl (like O-16:0) moiety and 2 Fatty Acyl moieties, and the user can choose whether to iterate over combinations of the 1 Plasmanyl moiety or the 2 Fatty Acyl moieties.

  • A Neutral Loss will only subtract the mass of that moiety from the whole lipid, and retain the adduct.

  • A Fragment will only be the mass of that moiety, and will become a molecular ion adduct like [M]+ or [M]-, losing the adduct.

Intensity, Charge state, Chemical Formula modifier, Fragment required for evidence level

  • Set the intensity between 1 - 999 indicating the relative intensity of the fragment peak in the MSn spectrum.
  • Set the Charge to the absolute value of the Charge. Charge cannot be 0 or a negative value.
  • Set the Chemical formula modifier if you need a change in the total formula that is smaller than a part of the lipid
    • This can be a gain chemical formula like C2H3O2 or a loss like H-2O-1, or a mixture of gain and loss like C2H3O2 H-2O-1
  • If you check the box for Fragment required for evidence level, then this indicates to downstream LipiDex processing that this fragment will be required to upgrade the ID from Sum composition to Molecular species.
    • For example, a PC in Sum composition would be PC 34:2 whereas the Molecular species level could be PC 16:0_18:2 or PC 16:1_18:1 or PC 14:1_20:1 or many other combinations.

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