PTM detection

Currently, Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) is the most widely used high throughput technique for analysing PTMs.^11,12. It is an analytical technique that combines the physical separation capability of liquid chromatography with the mass analysis capability of mass spectrometry. PTMs affect the molecular weight of the modified peptide and this differences in mass can be detected by MS/MS.

1.2.1. Liquid Chromatography Tandem Mass Spectrometry

Liquid Chromatography (LC) separates the components of a liquid mixture by percolating through a column. The velocity at which individual molecules in the mixtures move through the column is a function of the physical property of the analytes, the content of the column and the composition of the mobile phase. The time at which a specific analyte elutes from the column is called the retention time. Upon elution the analytes are directly sprayed into the mass spectrometer using electrospray, where the analytes are first ionized and then measured in the first mass spectrometer. The resulting spectrum provides the mass to charge (m/z) ratio of the intact analyte ions (precursor ions) that are coming off the LC. Because the precursor m/z cannot be used to uniquely define the complete structure of analytes, a second measurement is taken. The ions of a precursor are selected, and fragment ions are created and measured in the second mass spectrometer to produce the MS/MS spectrum for the analyte (Figure 1).

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Figure 1. Schematic of tandem mass spectrometry. The figure was originally published by Murray.¹³

Common fragmentation methods that are used for proteomics and glycomics are: collision induced dissociation (CID), higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD)^7,14. Historically CID has been the most commonly used, however HCD and ETD are getting more popular for both proteomics and glycomics. Especially for studying PTM’s^15,16. Figure 2 shows the backbone fragmentation locations and ion types for glycans and peptides. The ion types that are observed, depend on the fragmentation method that is used.

Figure 2. Ion types produced by collision fragmentation of (A) glycans and (B) peptide. The figures were originally published by Han⁷ and Roepstorff¹⁷.

A B

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The fragments in Figure 2 are not the only fragments that are observed in a MS/MS spectrum.

This is illustrated by Figure 3 which shows examples of a CID MS/MS spectrum for a glycan and a peptide. As can be seen, the spectrum contains peaks that cannot be assigned to backbone fragments. Possible sources of these peaks include neutral losses, side chain fragments and ions due to multiple backbone fragmentations.

Figure 3. Annotated CID MS/MS spectrum obtain from (A) glycan and (B) peptide. The figures were originally published by Han⁷ and Hernandez¹⁸.

1.2.2. Targeted PTM vs. open PTM proteomics

LC-MS/MS has become the standard tool for identifying PTMs of proteins. Figure 4 illustrates a general workflow for a proteomics experiment. It starts with the isolation of proteins from biological samples such as whole cells or tissue lysate, followed by enzymatically digesting of the protein into peptides. The most commonly used enzyme is trypsin which cleaves the protein after lysine or arginine residues, producing peptides averaging 15 amino acids in length¹⁸. These peptides are then measured in a LC-MS/MS and the resulting spectra are assigned to a peptide and then to a protein using bioinformatic tools.

B

A

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A typical MS/MS experiment results in spectra that can come from an unmodified peptide, a peptide modified due to sample preparation or a peptide carrying one or more modifications¹⁹. Unmodified spectra are generally analysed using a database search, where the experimental MS/MS spectra are matched against a user-selected protein database. Modified peptides can be analysed using a database search by specifying the modification prior to the search. This is called a targeted PTM search.

For a LC-MS/MS experiment where a targeted search was used, only about 25% of the spectra are expected to be identified²⁰. A large proportion of the unidentified spectra are likely to be peptides carrying modifications which were not considered in the targeted PTM search²¹. Including many modifications (>5) in a targeted search is not practical as it will dramatically increase the search time and reduce sensitivity¹⁹. Instead, a technique called open modification searches (OMS)¹⁹ is used. OMS do not require prior specification of the modification that are in the sample. Instead the modification is read out directly from the MS/MS spectrum allowing both known and novel PTMs to be identified.

Figure 4. Proteomics strategies for PTM analysis. (A) Targeted PTM analysis: The sample is enriched for the PTM of interest before being analysed using a database search. (B) Open PTM search: The sample is analysed using an open modification search. This allows both known and novel PTMs to be identified.

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1.2.3. Glycomics

Post translational modifications that have a more complex structure, such as glycans, cannot be identified using a targeted or open search. This is because the mass of these modifications is not enough to uniquely identify the chemical structure. For example, there are 5 different glycan structures that have a mass of 733²². Due to this ambiguity, different experimental and bioinformatics techniques are required to analyse glycans.

Figure 5 shows the general workflow of a glycomic experiment. First, proteins are isolated from biological samples, such as cell lines, tissue or body fluid. Then the glycans are either enzymatically (N-glycans) or chemically (O-glycans) released from these proteins. The released glycans are purified and then measured in a LC-MS/MS. The resulting MS/MS spectra are predominantly analysed by hand with the support of bioinformatics tools^23,24.

Figure 5. General workflow for analysing glycans by mass spectrometry.