Since the proof of principle of enhanced Ub- and SUMO-isopeptide analysis was established using the RUbI approach [54, 58], mTRAQ reagent  has been used to chemically derivatize the a-amino groups of the N-termini and iso-N-termini on a suite of both synthetic tryptic Ub-isopeptides and atypically tryptic SUMO(2/3)-isopeptides to further facilitate an enhancement in their analysis. This was shown to enhance qualitative analysis and introduce relative quantitation across three channels in both SWATH precursor and product-ion acquisition modes. Performance was demonstrated on a QTOF- based LC-MS/MS mass spectrometer with low-energy collision-cell CID, in a DIA SWATH MS approach termed mass spectral enhanced detection of Ubls using SWATH acquisition (MEDUSA) . Derivatization with mTRAQ imparts similar characteristic fragmentation behavior, product ion, and unique iso-N-terminal modification-specific product-ion generation on Ub- and SUMO(2/3)-isopeptides as dimethylation. However, the chemical composition and size of the mTRAQ modification combined with the presence of a carbonyl group within its structure assist in providing additional advantages over dimethylation by facilitating the generation of (i) additional N-terminal b1-type and diagnostic iso-N-terminal b1'-type product ions, (ii) more abundant diagnostic b'-type ions from the iso-N-terminus, and (iii) b-ions from the iso- N-terminus in a higher m/z range, which may benefit mass spectrometers where the transmission and observation of low mass ions is problematic. The advantages of improved product b' -type ion generation from the iso-N-terminus of the Ub/SUMO modification specifically combined with the ability of SWATH MS DIA to data capture all product ions from all detectable precursor ions in a single scan cycle, enables improved (i) comprehensive identification of the specific modification (ii) and enhancement toward the analysis of Ub- and SUMO-isopeptides, and (iii) provides greater quantitative selectivity of both Ub- and SUMO-isopeptides in SWATH product-ion acquisition mode. Figure 6.7 illustrates an overlay of the perfect coelution of the XICs of the diagnostic iso-N-terminal modification-specific a1' and b' ions required to indicate and subsequently enable comprehensive diagnostic identification. The most abundant b'-ion from each isotag confirms the presence of the full GG isotag representing a Ub modification or the full TGG and QTGG isotags representing the presence of a SUMO(2/3) modification on three different synthetic isopeptides.
Figure 6.7 Postacquisition extracted diagnostic a'-type and b'-type product ions generated during SWATH acquisition of the synthetic isopeptides with the backbone sequence, NSSYVLL(K}TGK bearing the following iso-chains on its internal lysine residue; (K), after labeling with mTRAQ 8. (a) GG iso-chin with a/ = m/z 178.1430, by = m/z 206.1379, and b2' = m/z 263.1594; (b) TGG iso-chain with a1' = m/z 222.1692, b1' = m/z 277.1750, and b3' = m/z 364.2070; and (c) QTGG iso-chain with a,' = m/z 249.1801, b/ = m/z 277.1750, b3' = m/z 435.2442, and b4' = m/z 492.2656. Source: Griffiths, 2014 . Reproduced with permission from Springer.
The use of triplex mTRAQ reagents - Д0, Д4, and Д8 - in the MEDUSA approach enabled the quantification of Ub- and SUMO(2/3)-isopeptides across three channels in the SWATH MS mode. The high-quality and the most abundant diagnostic b' product ion generated from the full iso-N-terminal Ub/SUMO(2/3) modification enables quantification in SWATH MS/MS mode. Quantification of synthetic Ub- and SUMO(2)/(3)-isopeptides using the MEDUSA approach was achieved by spiking two categories of predetermined theoretical ratios of these synthetic isopeptides - (i) 1(Д0):2(Д4):10(Д8) and (ii) 1(Д0):5(Д4):10(Д8) - into a background of Escherichia coli peptides, which had also been labeled at a predetermined theoretical ratio of 1(Д0):1(Д4):1(Д8). Figure 6.8 shows an example of the quantification of both Ub-isopeptides. Figure 6.8a is an overlay of the three integrated and extracted monoisotopic precursor ion chromatograms from the TOF MS, which represent the three channels the synthetic Ub-isopeptide had been spiked in to the E. coli background at a known ratio of 1(Д0):5(Д4):10(Д8). The calculated peak areas for each of the three peaks enabled the relative quantification of a ratio that was equivalent to 1(Д 0):5.3(Д 4):10.7(Д8), with a high degree of accuracy in comparison to the predetermined theoretical ratio. The same Ub- isopeptide at the same known ratio was quantified in the SWATH-MS/MS product-ion acquisition mode, Figure 6.8b is an overlay of the three integrated and extracted dominant diagnostic b2' ion representing the GG isotag from each of the three channels; m/z 255.1452 (Д0), m/z 259.1523 (Д4), and m/z 263.1594 (Д8). The calculated peak areas for each of the three peaks enabled the relative quantification with a ratio equivalent to 1(Д0):6.6(Д4):18.8(Д8) to a lower degree of accuracy in comparison to the predetermined theoretical ratio. The MEDUSA approach successfully enabled a suite of synthetic Ub- and SUMO(2)/(3)-isopeptides to be relatively quantified across three channels using (i) the extracted monoisotopic precursor ion peak areas from the SWATH-MS spectra or (ii) the most abundant diagnostic product-ion peak areas from the SWATH-MS/MS spectra. As expected, the MEDUSA approach has the same minor caveat to the RUbI approach in that the iso-N-terminal diagnostic ions would also be generated from tryptic linear peptides with a GG at their N-terminus; however, experimental and theoretical agreement is that this type of false-positive result would occur at a rate of 0.5% [54, 60]. Depending on the prevalence of N-terminal protein ubiquitination, which occurs significantly less frequently than lysine ubiquitination, the percentage of 0.5% can be further reduced due to the superior b- and b'-type product-ion coverage generated from m-TRAQ-labeled isopeptides in comparison to RUbI, enabling us to distinguish between two isomeric species, a false-positive
Figure 6.8 MS and MS/MS-based quantitation of isopeptides with the backbone sequence, EGV(K)TENNDHINLK bearing the GG iso-chain on its internal lysine residue; (K), based upon extracted ion peak areas. Of the three co-eluting extracted monoisotopic precursor ion chromatogram traces on (a) and (b); the trace of lowest intensity corresponds to ДО-tagged (100 fmol), the trace of mid-range intensity relates to ДТ-tagged (500 fmol), and the trace of highest intensity represent Дв-tagged (1 pmol) isopeptides. Source: Griffiths, 2014 . Reproduced with permission from Springer.
linear peptide with a GG at its N-terminus and its equivalent Ub-isopeptide. This was possible due to the presence of a set of a/a'- and b/b'-type production pairs in the MS/MS spectrum. This set of product-ion pairs was absent from the MS/MS spectrum of the linear peptide containing an N-terminal GG, which only contained the a- and b-type product ion with further consecutive b-type product ions from the backbone of the linear peptide confirming the remainder of its N-terminal sequence .