Home Health Analysis of Protein Post-Translational Modifications by Mass Spectrometry
Mass Spectrometry Behavior of Sulfated Peptides
Tyrosine sulfation introduces a strongly acidic modification to the peptide. One would expect that this modification negatively influences the mass spectrometry response in positive ion mode and lowers the number of charges deposited on the modified peptide by electrospray ionization. From this angle, sulfopeptides resemble phosphopeptides. In addition, both modifications increase the peptide mass by nominally 80 Da: the mass difference between these isobaric structures is only 9 mmu (the exact additive masses of the modifications are 79.9663 Da and 79.9568 Da for phosphate and sulfate, respectively). What makes the sulfopeptides unique is that this modification is more fragile than phosphorylation, and a partial neutral loss of SO3 is regularly observed upon mass measurement of the intact peptide (Figure 9.1, inset),
Table 9.1 Reliably assigned human sulfo-Tyr sites from UniProt May 2015.
while gas-phase desulfation is 100% upon collisional activation (Figure 9.1). This fragmentation step is favored so much that in ion trap collision-induced dissociation (CID), that is, under resonance-activation conditions, usually the only fragment observed is the product of desulfation (data not shown).
Mass spectrometry analysis of a sulfopeptide has been described as early as 1987: an a2-antiplasmin peptide was analyzed using fast atom bombardment
Figure 9.1 Beam-type CID (higher-energy C-trap dissociation (HCD) in the Thermo nomenclature) spectrum of m/z 937.3632(2+) corresponding to
(FAB) ionization in positive ion mode, and the detection of the intact peptide, its sodium adduct, and the sulfate loss yielding an abundant unmodified peptide ion have been reported . Later, it became obvious that gas-phase sulfate loss (-80 Da) is a characteristic feature of sulfopeptides: in positive mode, the modification may completely disappear, but desulfation may occur even in negative ion mode . The extent of this gas-phase desulfation is influenced by the ionization method: under normal MALDI conditions, sulfopeptides require analysis to be performed in negative mode, while the gentler atmospheric pressure MALDI permits sulfopeptide detection even in positive mode . We postulate that the same results could be achieved with regular MALDI-TOF instruments using 2,6-dihydroxyacetophenone as the matrix  as for equally fragile phospho-His-containing peptides . Larger polypeptides may retain the modification in positive linear mode even in conventional matrices .
Unfortunately, even the gentler electrospray ionization may trigger abundant SO3 losses [31, 38, 49]. MS acquisition conditions have to be adjusted to pre- vent/minimize this phenomenon in the mass measurement experiments. For example, lowering the temperature of the heated capillary in certain mass spectrometers significantly eliminates gas-phase desulfation and, thus, increases the sulfopeptide signal . Unfortunately, while the sulfate loss may be minimized in the MS surveys, collisional activation leads to complete elimination of the modification. Thus, the peptide fragments allow the amino acid sequence assignment; however, the site localization is impossible from CID data [31, 38]. At the same time, Wolfender et al. presented high-energy CID data in negative mode where the diagnostic fragment ions for sulfation (m/z 80) and for sulfo- Tyr (m/z 214) were detected .
An obvious solution for assigning fragile modifications to precise amino acids is the application of electron-transfer dissociation (ETD). Indeed, Mikesh et al. demonstrated that the method may work for sulfopeptides, albeit the synthetic peptide analyzed was modified on a Ser residue . However, more frequently than not, sulfation works against forming a precursor ion of sufficient charge density. One can expect that most tryptic sulfopeptides will be detected as doubly charged ions. Doubly charged precursor ions may produce sufficient information for modification site assignment as illustrated by Figure 9.2. At the same time, in most cases - unless a peptide features “reasonable” charge density - the resulting ETD spectra will not be informative enough even for peptide identification , with longer or multiply modified sulfopep- tides most likely fitting this category.
Since sulfopeptides form negative ions with relatively high efficiency, especially with multiple modifications, some research groups turned to this direction when searching for alternative solutions. Hersberger and Hakansson described sulfopeptide fragmentation in negative ion CID, electron detachment dissociation (EDD), negative ETD (NETD), and negative ion mode ECD (niECD) . In EDD, radical, charge-reduced ions are formed using a high-energy electron beam hitting multiply charged negative ions. Fragmentation occurs along the peptide backbone, between the a-carbons and the carbonyl groups, resulting in a* and x-ion formation, and, crucially, leaving the side chains intact. In the negative variation of ETD (NETD), the multiply charged anion transfers an electron to an acceptor molecule, eventually leading to the same type of fragmentation as in EDD. These methods suffer from the same problem as ETD: producing high charge density negative ions is more difficult than achieving the same in positive mode. Thus, these methods do not offer real solution. On the other hand, niECD produces charge-increased precursor ions via triggering electron capture by irradiating peptide anions with lower-energy electrons. In these multiply charged peptide radicals, the fragmentation occurs between the amino group and the a-carbon, producing primarily c' and z* ions, while retaining the sulfate modification (Figure 9.3). While this method may yield more extensive sequence cover-
Figure 9.2 ETD spectrum of m/z 937.3632(2+) corresponding to
age for sulfopeptides than ETD, due to its low sensitivity and efficiency, its utility as a truly viable alternative is questionable.
Using another alternative technique, Robinson et al. demonstrated that 193 nm ultraviolet photodissociation (UVPD) generates an informative MS/ MS spectrum from sulfopeptide anions. The results are similar to ETD fragmentation in that while sulfate loss is regularly detected from the precursor and charge-reduced precursor ions, the fragments usually retain the modification (Figure 9.4) . Unfortunately, this method is not sensitive enough for routine LC-MS/MS analysis either.
Chemical derivatization has been used for improving/controlling MS/MS fragmentation as well as for enabling the site assignment of other elusive PTMs. Yu et al. used a clever “trick” for finding sulfated residues in purified proteins: the unmodified Tyr residues were acetylated using sulfosuccinimidyl acetate (S-NHSAc) in the presence of imidazole at pH 7.0 . This way even
Figure 9.3 NiECD data of sulfo-hirudin, the precursor ion was singly charged.
Source: Hersberger, 2012. . Reproduced with permission from American Chemical Society.
Figure 9.4 Negative UVPD mass spectrum (three pulses at 2 mJ) of bovine fibrinopeptide B, (2-) from the average of 18 MS/MS scans acquired over 12 s. SO3 neutral loss products are annotated in light grey. GIp stands for pyroglutamic acid. Source: Robinson, 2014 . Reproduced with permission from Springer.
the gas-phase desulfation does not prevent the accurate site assignments since detected tyrosine residues without the modification are assumed to be sul- fated (Figure 9.5).
Figure 9.5 Identification of the site of sulfation in lumican peptide 29Met-Tyr52. The unmodified Tyr residues as well as the N-terminus were derivatized with sulfosuccinimidyl acetate. Thus, the only "unmodified" Tyr represents the modification site. Source: Yu, 2007. , Reproduced with permission from Nature Publishing Group.
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