Experimental set-up for PTM identification

Studying the results presented in the papers describing the OMS tools discussed above it becomes clear that the vast majority of the modifications identified are in fact not post-translational but rather chemical modifications induced by sample preparation such as Cysteine Carboxyamidomethylation, N-terminus and Lysine Carbamylation, Oxidation of Methionine and Sodium and Potassium adducts. PTMs especially those previously unknown can be expected to be poorly abundant. This is illustrated in Figure 5 showing the modification mass distribution of a typical OMS search. The histogram displays the confident PSMs returned when screening a human blood plasma sample dataset produced on an Orbitrap instrument, and analysed with a novel library search based OMS tool, QuickMod (Ahrné et al. manuscript in preparation).

The detection of low-abundant post-translationally modified peptides is very limited when analysing complex mixtures because these peptides are overshadowed by unmodified peptides and peptides modified during the sample preparation. In addition, post-processing algorithms tend to favour the identification of abundant modifications for which extensive evidence can be found. These factors complicate the successful discovery of rare PTMs. The detection problem can be partly improved by various promising sample preparation techniques such as anti-phosphoamino acid antibodies for protein isolation [83, 7] and affinity-based enrichment of modified proteins or peptides [6,84-85].

Seo et al. present a protocol targeting low abundance PTMs [86]. The authors describe a clever LC-MS workflow, Selectively Excluded Mass Screening Analysis (SEMSA) where samples are analysed by an LC-ESI-qTOF in multiple rounds. For each round, the precursor masses of spectra confidently identified, using MODi [55], are added to a mass exclusion list

allowing for the fragmentation of precursor ions of low intensities. A similar set-up, presented in Schmidt et al. [87] where unidentified MS features were added to an inclusion list for targeted fragmentation, lead to extensive identification of phosphorylation sites in a protein mixture obtained from Drosophila melanogaster lysates.

Another interesting LC-MS identification workflow was recently published by Carapito et al. [88] where spectra are acquired under different collision conditions and a peptide mass inclusion list is compiled based on the detection of modification specific neutral loss fragments and reporter ions in combination with ion signals corresponding to the modified and unmodified peptide masses. In a final step the peptides on the mass inclusion list are sequenced in a directed MS/MS mode.

Barnes et al. [89] developed a software tool, MassShiftFinder, tackling the detection problem by screening MALDI-TOF data for potentially modified peptides which then can be selected for subsequent TOF-TOF analysis. Their algorithm performs a blind search for modifications using peptide mass fingerprints from two proteases with different cleavage specificities. If the same mass shift relative to the unmodified theoretical values is observed for both proteases, and the peptides are overlapping, the mass shift can correspond to a modification or a substitution.

Working with more than one proteases is in general a good idea in order to increase the sequence coverage of PTM sites in the results of analysis [10]. Strong b- or y- ion peaks on either side of a modified residue are the best evidence for site specificity. Digesting the samples with multiple proteases also improves the chances of producing such a spectrum, facilitating the modification localisation problem. Furthermore, the confident identification of low abundance peptides generally requires multiple replicate analyses of the same LC-MS/MS of similar or replicate samples [65].

As mentioned earlier in this review, an additional problem which makes the identification of real PTMs particularly tricky is the fact that some modified peptides fragment poorly in the mass spectrometer. Low-energy CAD/CID tandem mass spectrometry has been, by far, the most common method used to dissociate peptide ions for subsequent sequence analysis. Ideally, the peptide is cleaved randomly at the amide bonds along its backbone to produce a homologous series of b and y-type fragment ions. The presence of multiple basic residues prevents full fragmentation upon collision activation/induction and

112 directs the backbone bond dissociation to specific sites and therefore inhibits the production of a sufficiently diverse set of sequence ions. Further, post-translational modifications such as phosphorylation, sulfonation, nitrosylation and O- and N- linked glycosylation may similarly redirect the sites of preferred cleavage. Often the modified moiety is cleaved off and the peptide backbone is left more or less intact. The resulting spectra tend to contain little peptide sequence information and may not allow for successful identification. In this regard, CAD/CID is most effective for short, low-charged unmodified peptides. New instrumentation technologies support alternative solutions for data generation that have the potential to improve peptide and protein identification, in particular the identification of peptides carrying labile modifications.

As n-dimensional mass spectrometry has become more practical new techniques to identify modified peptides have been developed making up for the limitations of CAD/CID fragmentation. Newer ion-trap instruments provide the option of collecting MS3 spectra of abundant MS2 peaks. Peptides carrying labile modifications have been analysed by automated data-dependent triggering of MS3 acquisition whenever the dominant neutral loss ion of the appropriate mass is detected in an MS2 spectrum [8, 90, 91]. By separately fragmenting the neutral loss ion a sequence information rich MS3 spectrum can be produced. Different approaches have been tested to combine MS2 and MS3 spectra from the same peptide to improve peptide identification [92-94].

Other methods to generate higher quality spectra of peptides carrying labile modifications rely on new fragmentation techniques altogether. Electron Capture Dissociation (ECD), is a method for peptide dissociation which is relatively indifferent to peptide sequence and length while avoiding the loss of labile modifications during fragmentation [95].

However, ECD requires an FT-ICR mass spectrometer. Syka et al. [96] introduced Electron Transfer Dissociation ETD and has proven useful for the identification of modified peptides and peptides with basic residues. ETD fragments peptides at the Cα-N bond by transferring an electron from a radical anion to a protonated peptide inducing similar fragmentation patterns to ECD, but can be used on more widely accessible ion-trap or Orbitrap mass spectrometers [97]. Olsen et al. [98] demonstrated a third new PTM-friendly fragmentation technology which takes advantage of the Orbitrap's architecture; Higher energy C-trap dissociation (HCD). HCD spectra show richer fragmentation than typical CAD/CID spectra especially in the low-mass region of the spectrum including a2, b2, y1, y2 ions and immonium ions of histidine and modified residues such as the immonium ion of phosphotyrosine.

Many more experimental protocols have been described in the literature aiming to increase identification of PTMs. For details we refer to an excellent review on this topic [3].

Figure 5) The vast majority of the modifications identified in a typical OMS are in fact not Post-Translational but rather modifications induced by the sample preparation such as Cysteine Carboxyamidomethylation, N-terminus and Lysine Carbamylation, Oxidation of Methionine and Sodium and Potassium adducts. PTMs especially those previously unknown can be expected to be low abundant. The histogram display the confident modified PSM returned when screening a human blood plasma sample dataset produced on a Orbitrap instrument, and analysed with a novel library search based OMS tool, QuickMod (manuscript in preparation).