3.1 General comments
The basic analytical strategy consists in gathering a maximum of structural information by obtaining molecular weight data on the intact protein(s) and then dissociating the molecular species to generate complementary ion pairs that have sufficient abundances and appropriate masses for subsequent further stages of tandem MS. In theory, given that the LTQ-Orbitrap offers three potential sites for ion activation (the nozzle-skimmer region, the linear ion trap (LTQ) and C-trap), multiple sequential MSn
experiments should be feasible (see supplementary information
). However, this scenario is not fully realizable in practice due to the significant losses in ion abundance that occur during each MS/MS step. For the Orbitrap Discovery, we have found the most practical approach to be the use of nozzle-skimmer dissociation (NSD) to yield a rich array of MS2
fragment ions that can be subsequently sampled by mass selection and collision-induced decomposition in the LTQ, followed by high resolution, accurate mass determinations of the fragments in the Orbitrap. Although the C-trap can be used for MS/MS with HCD, our results have so far demonstrated a significant loss in signal intensity when using this activation method. (Upgrade to Velos-type optics may partially or fully address this loss of sensitivity. Furthermore, although ETD can also be carried out on the LTQ-Orbitrap [34
], this option had not yet been installed on our instrument when the reported analyses were carried out. It will be evaluated in future experiments.)
The sequence information obtained by NSD followed by LTQ-CID of the resulting fragment ions can be extended or confirmed by LTQ-CID of mass-selected individual charge states of the intact protein. For the two proteins that are the subject of this study, only minimal sample preparation was needed to achieve adequate sensitivity for clinically relevant sample amounts in analyses based on nozzle-skimmer dissociation for with. TTR can easily be isolated in a pure state by immunoprecipitation from serum; hemoglobins consist of a simple mixture of two proteins that can both be clearly observed by ESI-MS upon 500-fold dilution of whole blood.
A preliminary mass profile of each sample was first generated to determine the precise mass shift that could be attributed to an amino acid substitution or post-translational modification (PTM). This information enabled identification of the NSD-generated fragment(s) that bear an amino acid substitution or PTM. The relevant fragment ion(s) could, individually, be selected for MS3 analysis to provide more detailed structural information. In the application of this method, it should be noted that, even in the absence of an apparent mass shift the NSD spectrum should always be acquired, given the possibility of variant mass shifts too small to be noted in the mass profile or of double sequence variations that could compensate one another, thus giving rise to small net mass shifts. The inspection of sequence-defining complementary ion pairs should assure the detection of all mass shifts, even those that are small or serve to compensate one another.
In development of the method, the next step was to mass-select specific intact protein charge states to map out the fragmentation of the analyte(s). It is of paramount importance to ascertain the origin of the NSD fragment ions if NSD/LTQ-CID MS/MS is to be used for sequence analysis. In the case of hereditary disorders, unless the sample donor is a homozygote, both wild-type and variant proteins can be detected. Furthermore, extensive PTMs are usually present in TTR and these give rise to multiple isoforms of both the wild-type and variant protein [24
]. The fragmentation behavior of each of the major post-translationally modified forms was investigated to assure that these modifications could be determined. The hemoglobin samples, although much less subject to post-translational modification, consist of both α and β chains and this system offers its own challenges in the implementation of the NSD/LTQ-CID MS/MS analytical scheme.
It is well known that the fragmentation of proteins varies over the different charge state isoforms selected for Top-down analysis (charge state-dependent dissociation behavior) and Mekecha et al.
] have used this observation to generate extensive α-hemoglobin sequence coverage by recording the fragmentation of the full range of the protein charge states from 4+ to 20+ using a modified ion trap. However, this approach is not consistent with the methodology for higher throughput analysis of variant sequences developed herein for use in a clinical setting.
3.2 Top-Down analysis of transthyretin
As noted above, there is no previous report of a Top-down analysis of transthyretin, apart from our published study of TTR fragments that ranged from 7 to 10 kDa that were extracted from amyloid fibrils. Of the two analytical challenges considered in this paper, the characterization of TTR variants presents the simpler case, despite the presence of extensive PTMs. shows the nanoESI mass spectrum of a wild-type TTR sample; the inset provides an expansion of the region that includes the 14+ charge state of the molecular-weight related species. The TTR post-translational modifications overwhelmingly center on Cys 10, the only cysteine residue of the protein, and generate the S
-thiolated (cysteine, CysGly and glutathione) and S
-sulfonated forms [24
]. The ratio between the two most abundant post-translationally modified forms, S
-cysteinylated and S
-sulfonated TTR, can vary widely from sample to sample. A higher abundance of S
-sulfonation with respect to S
-cysteinylation has been speculated to reflect disease states [36
]. The S
-CysGly and S
-glutathionylated forms of TTR are present in low abundance. The unmodified form of the protein typically represents less than 15% of the total TTR-related isoforms observed in the mass spectra of TTR immunoprecipitated from human serum. One factor of note is the differernce in the stabilities of these post-translational modifications under collisional activation conditions typically employed for NSD and CID. The S
-thiolated forms are quite stable and generally remain intact. On the other hand, the modification in S
-sulfonated TTR is very labile; the S
-sulfonate group on a cysteine residue is easily cleaved [38
] and the released protein appears in the NSD mass spectrum as unmodified TTR. However although this post-translational modification is lost as a result of the activation method, the capability of the method to obtain the complete amino acid sequence information for variant characterization is not adversely affected.
NanoESI mass spectrum of a wild-type TTR immunoprecipitated from serum. The inset provides an expansion of the region that includes the 14+ charge state (m/z 980–1010) and the various post-translationally modified forms of TTR.
As discussed above, the most common approach to Top-down MS/MS analysis of proteins is to mass select individual charge states of intact protein ions for MS/MS. The LTQ-CID MS/MS spectrum of the most abundant charge state, [M + 14H]14+ m/z
992.43, of a post-translationally modified wild-type TTR is shown in . This ion corresponds to the S
-cysteinylated form of TTR. This mode of fragmentation does not yield extensive sequence coverage. The core section of the sequence and the N-terminal region are not very well covered. Fragmentation is dominated by the sequence-defining complementary ion pair b42
, as well as a series of low mass y-ions describing the C-terminus that cover positions 117 to 125 and tend to be ubiquitous features of Top-down spectra of TTR-derived proteins using CID [29
]. The high abundance of the C-terminal fragments is well illustrated by the expansion factor (×3) necessary to visualize the other fragments in . Higher collision energies favor the appearance of this ion series above all others.
Figure 2 (a) Deconvoluted LTQ-CID MS2 spectrum of m/z 992.43 corresponding to the [M + 14H]14+ of S-cysteinylated wild type TTR. (b) Since the S-cysteinyl post-translational modification is eliminated immediately upon CID, the sequence coverage generated by BUPID (more ...)
More complete initial coverage can be obtained by interrogating several different charge states (data not shown). It should be noted that the use of higher skimmer potentials in NSD can lead to proton stripping. This can be advantageous for the Top-down approach, as it gives the analyst access to lower charge states in significant abundance, enabling further probing of the charge state-dependent dissociation behavior. For example, an abundant TTR8+
charge state is generated at higher skimmer voltages and can be subjected to MS/MS. Dissociation of this charge state showed suppression of the extensive C-terminal low mass fragmentation which, as noted above, tends to dominate the MS/MS spectrum of most charge states of the TTR isoforms and thus yielded broader sequence coverage (supplementary data
). This observation can probably rationalized on the basis of the proton mobility fragmentation model as the TTR sequence contains 10 basic sites. Presumably, in the lower charge states, a proton is less likely to be in a position to initiate fragmentation of the C-terminal region than in more highly charged isoform of the protein. In other words, charge might be sequestered away from the C-terminal activation site in the TTR8+
isoform. Important questions remain to be explored in the study of protein CID mechanisms [40
] but it is beyond the scope of this paper to address them.
MS3 analysis of the very abundant b42/y85 pair generated by CID of a selected charge state is possible and could be used for variant analysis. Nevertheless, despite the potential illustrated by precursor ion selection and fragmentation in the LTQ, our further investigations indicated that nozzle-skimmer dissociation offered greater advantages, in terms of both experimental simplicity and observed sensitivity, as the first step in the Top-down analysis of TTR. The fact that the immunoprecipitation step used as the isolation procedure for transthyretin yields the protein in a very pure state, whereby no interference is present upon MS analysis, allows the direct use of NSD. A representative NSD MS2 fragment ion spectrum of immunoprecipitated TTR is presented in .
(a) Deconvoluted NSD MS2 spectrum of wild type TTR immunoprecipitated from serum. (b) Sequence coverage generated by BUPID.
The mass spectrum of this sample indicated that S-cysteinylation was the most abundant post-translational modification. The most notable feature of this spectrum is the presence of the sequence-defining complementary ion pair, b42/y85. These two fragment ions capture the complete 127-amino acid TTR sequence. The genesis of this pair can easily be rationalized as it arises via the highly favored cleavage of a glutamic acid/proline (Glu42-Pro43) peptide bond. Of note is the diminished abundance of the low mass y-ion series, as compared to that found in the LTQ-CID of the [M + 14H]14+ m/z 992.43 shown in . For this type of experiment, a lower fragmentation voltage could is selected in order to favor the formation of an abundant b42/y85 complementary ion pair for subsequent LTQ-CID MS3. The presence of a relatively abundant internal ion I43–63, resulting from cleavages of both the Glu42-Pro43 and Glu63-Phe64 bonds probably results from secondary fragmentation of the y85 fragment ion. Such internal ions can arise from secondary cleavages of b- or y-ions. These species can be identified by BUPID (Although they were not needed for interpretation of the data presented herein, failure to properly assign them could introduce ambiguity to the sequence analysis.). At this point, the reliability of the assignment using b- and y-ions is the most important factor to be considered and, for that reason, we have chosen not to discuss internal fragment ions save for exceptional cases. As indicated in the spectrum shown in , the [y85 + 9H]9+ fragment ion (m/z 1039.861) is produced in high abundance even at relatively low “fragmentation” potential. The MS3 spectrum of this NSD-generated fragment ion was produced by CID of the selected precursor in the LTQ and mass analysis of the products in the Orbitrap, which yielded a substantial set of accurate mass values for the b-, y- and internal fragments; the most abundant of these are labeled on the spectrum shown in . The coverage obtained in this manner was specific and extensive, as indicated in the scheme that is presented above the spectrum.
Figure 4 (a) Deconvoluted LTQ-CID MS3 spectrum of the [[y85 + 9H] 9+ fragment ion, containing residues 43-127, generated from the NSD of wild-type TTR immunoprecipitated from serum. The sequence coverage generated by BUPID is shown above the spectrum. (b) Deconvoluted (more ...)
For TTR, we have determined that the [y33 + 3H]3+ (m/z 1198.279 for wild-type TTR) and [y45 + 4H]4+ (m/z 1224.619 for wild-type TTR) fragment ions also yield to detailed structural information on the C-terminal portion of the protein. Only positions 100–110 did not exhibit definitive fragments in any of the modes utilized to gather sequence information on this region of the protein. Using an FT-ICR instrument, we have recently determined that coverage of this region of the sequence can be improved by the use of electron transfer dissociation (ETD) or electron capture dissociation (ECD). These additional approaches will be explored further and the results will be reported in a subsequent contribution.
Obtaining an informative MS3
spectrum for the cysteinylated [b42
fragment ion observed at m/z
906.060 () was more challenging, given the heterogeneity introduced by the Cys10-based PTMs [24
] that distributed the signal over multiple species, and the lower absolute yield of this fragment ion. The coverage obtained in the MS3
spectrum for the cysteinylated [b42
fragment ion was extensive, with a conspicuous b18
ion pair resulting from a cleavage on the C-terminal side of an aspartic acid residue.
Although the abundance of fragment ions could be moderately increased by varying the skimmer potential, the abundance of the b42 species remained lower than that of the y85 ion at any given value of this parameter. Nevertheless, these factors did not significantly hinder obtaining N-terminal sequence coverage from MS3 of the [b42 + 5H]5+ ion, even though they decreased the numbers of ions available for that purpose.
For an unknown or partially known protein, subjecting each member of a complementary ion pair, such as this b42/y85 example, to further tandem MSn analysis can quickly yield substantial sequence information. As shown here in the case of variant sample analysis, the full sequence of the wild-type protein has already been established and, thus, the selection of the proper member of the complementary ion pair for further dissociation depends solely on the determination of which of the two fragments exhibits the same mass shift as that observed in the previously recorded mass profile of the intact protein sample. The variant fragment ion can then be mass selected for MS3 analysis to obtain sequence information.
The usefulness of the approach presented here can be appreciated if one considers the case of a Cis double mutation of Ser6 and Ala30 reported to have been detected by the combination of molecular weight profiling by mass spectrometry and DNA analysis [28
]. Using the method we describe here, mass spectrometry alone would be sufficient to detect this combination of changes in the protein structure. Examination of the b42
complementary ion pair in the expected NSD spectrum would reveal the presence of a +2-Da shift in the b425+
ion. No single amino acid substitution can give rise to a mass shift of +2 Da, so this data would indicate that a double mutation is likely present. Hence, the NSD data can increase the ability of the analyst to accurately measure small mass shifts due to single or multiple sequence variations. The MS3
analysis would include LTQ-CID of the of the b425+
ion, providing the added structural information neded to fully define the variant positions.
The approach described above was applied to a TTR sample immunoprecipitated from patient serum. The nanospray mass spectrum indicated the presence of an unknown variant with a +30-Da mass shift (). The NSD spectrum showed a peak having a 5+ charge at m/z 912.546 that corresponded to a shift of +30 Da for the cysteinylated b42 fragment of the variant vs. that of the cysteinylated wild-type protein, and thus indicated that the variant was present within the first 42 positions of the sequence ().
Figure 5 NanoESI mass spectrum of an unknown TTR variant immunoprecipitated from serum exhibiting a +30-Da mass shift. The inset provides an expansion of the region that includes the 14+ charge state (m/z 980–1010) showing the peaks representing the wild-type (more ...)
Figure 6 (a) Deconvoluted NSD2 mass spectrum of an unknown TTR variant immunoprecipitated from serum. The cysteinylated [b42 + 5H]5+ fragment is accompanied by a +30-Da component corresponding to the mass shift observed in the mass profile of the intact protein, (more ...)
No ion corresponding to (y85
+ 30) Da was observed. The position of the mutation could actually be narrowed somewhat by careful examination of the NSD data, following the b-ion series until the +30-Da mass shift was no longer observed. In this case, a b25
+ 30)-Da pair was observed, indicating that the amino acid substitution is in the first 25 amino acid positions of the sequence. The isotopic cluster at m/z
912.546, corresponding to the cysteinylated ([b42
+ 5H+ 30 Da]5+
) fragment ion, was mass selected for CID-MS/MS in the LTQ (). The fragmentation was compared to that obtained for the corresponding peak in the NSD spectrum of wild-type TTR. There was no change observed in the b-ion series, i.e
., the +30-Da mass shift was present in the smallest observed b-ion (b9
) and all higher members of this series, confirming that the amino acid substitution was present within the first nine positions of the sequence. The complementary y-ion series did not exhibit a +30-Da mass shift until y37
, indicating that the amino acid substitution was on Gly6 or Glu7 (see ). A +30-Da mass shift can only result from one of five possible amino acid substitutions (Gly→Ser, Ala→Thr, Thr→Met, Val→Glu, Arg→Trp), so this sequence change must be Gly6→Ser6The Gly6Ser variant is an established non-amyloidogenic polymorphism present in about 12% of humans [42
]. Although this data is diagnostic for the presence of a non-symptomatic variant rather than an instance of the genetic TTR amyloid disease (familial amyloidotic polyneuropathy), this example demonstrates the simplicity of the approach. In essence, our results indicate that the NSD MS2
spectrum, the subsequent MS3
CID of the appropriate NSD-generated fragment(s), and possibly the MS2
spectrum of the [M + 8H]8+
intact protein charge isoform should be analyzed together to generate maximum sequence coverage after obtaining a mass profile of the protein.
Neutral masses of the y-ion series obtained from the deconvolution of the LTQ-CID MS3 spectra of the b425+ fragment ion of the wild type and Gly6Ser variant.
3.3 Top-Down analysis of hemoglobins
Normal adult hemoglobins are composed of tetramers, each containing four globin chains. In normal adults, the most common components are two alpha (α) and two beta (β) chains. The α (Mr 15126.4) and β (Mr 15867.24) chains consist of 141 and 146 amino acids, respectively. The fact that the circulating hemoglobin tetramer consists of more than one protein complicates the challenge of elaborating a simple Top-down methodology. The option of separating the α and β chains from each other before MS analysis defeats the purpose of designing a simple approach, as two key assets of mass spectrometry are its speed and selectivity. Introducing a separation step would negate speed and ignore selectivity. Our goal in the experiments reported herein was to establish that the direct Top-down analysis of hemoglobin from whole blood can provide rapid extensive sequence coverage with minimal sample preparation and without chromatographic separation.
The LTQ-CID fragmentation obtained by dissociation of the abundant charge states for the α (15+) and β (17+) chains provides a wealth of information and appears to be more extensive than that obtained for TTR. The fragmentation of intact hemoglobin chains under collisional activation conditions has been described [8
] and our results are in broad agreement with reported observations. The fragmentation exhibited by the α chain is particularly rich and plentiful, as exemplified by the MS/MS spectrum of [M + 15H]15+ m/z
1009.408 (). The most abundant product ions are derived from facile cleavage at sites on the C-terminal side of acidic residues (b75
) and the N-terminal side of Pro (y28
). The fragment ion b75
results from the Asp75-Met76 cleavage C-terminal to aspartic acid. Interestingly, the y66
fragment which forms a complementary ion pair with b75
is reported [35
] to be abundant but is only observed with low signal intensity in this spectrum, with b61
being the closest-in-mass abundant fragment. The most dominant fragmentation channel is the cleavage at Leu113-Pro114 that gives rise to the y28
fragment. Similarly, the complementary ion in this pair, b113
, is only observed at low signal intensity, with b111
being the closest-in-mass abundant fragment. Many observed peaks could be assigned to products resulting from fragmentation in the “core” region of the protein and good overall sequence coverage was obtained, as shown in .
(a) Deconvoluted LTQ-CID MS3 spectrum of [M + 15H]15+ m/z 1009.408 from hemoglobin α chain in diluted whole blood. (b) Sequence coverage generated by BUPID.
In the LTQ-CID MS2
data obtained for dissociation of the [M + 17H]17+
of the β chain that was observed at m/z
934.324 (), the main feature of the MS/MS spectrum is the [y96
fragment arising from the facile cleavage of the Thr50-Pro51 peptide bond. The high mass y-ions y98
form complementary ion pairs with the b50
ions, respectively. Of note is the presence of a long series of b-ions, from b4
, describing most of the N-terminal sequence of the β chain, with the series from b30
being particularly prominent. These general observations are in qualitative agreement with those of Schaaff et al.
]. The core sequence information obtained from the LTQ-CID MS2
spectrum of the [M + 17H]17+
of the β chain is less extensive than was found for dissociation of the intact α chain [M + 15H]15+
ion (). These preliminary LTQ-CID MS2
spectra provided a preview of what could be expected from nozzle-skimmer dissociation of the α and β chains, and established the provenance of the fragment ions generated by NSD.
(a) Deconvoluted LTQ-CID MS2 spectrum obtained for the [M + 17H]17+ of the hemoglobin β chain in diluted whole blood that was observed at m/z 934.324. (b) Sequence coverage generated by BUPID.
The NSD spectrum of diluted whole blood is shown in . Many fragment ions can be observed. It proved possible to select a number of fragment ions from each chain that could be used to implement the strategy presented above for TTR, whereby MS3 analysis of complementary ion pairs is used to obtain complete sequence information.
Figure 9 Deconvoluted NSD MS2 spectrum of diluted whole blood. Most of the abundant peaks can be assigned as fragments of α or β hemoglobin. The key fragment ions constituting complementary ion pairs that could be used for subsequent LTQ-CID MS (more ...)
All the candidate fragment ion assignments were confirmed by MS/MS. In the case of the β chain, the prominent [y96
+ 11H]11+ m/z
940.42 and [b47
+ 5H]5+ m/z
1047.56, covering 143 of 146 amino acid positions, were determined to be suitable, as both fragment ions have reasonable abundance and are amenable to further MSn
they provide good sequence coverage/information). The y9611+
fragment ion could be efficiently generated in high abundance by NSD of the diluted blood sample and mass selected to undergo MS3
in the LTQ (). The sequence coverage thus obtained () provided information from the “core region” not available from the LTQ-CID mass spectrum obtained for the 17+
charge state of the β chain. This region of the β-chain sequence (residues 58-111) has been reported to be difficult to sequence using CID of the intact β chain [45
], but the approach presented here offers a simple and elegant solution to this problem.
(a) Deconvoluted LTQ-CID MS3 spectrum of the Hbβ [y96 + 11H]11+ fragment at m/z 940.42, containing residues 51-146, generated by NSD of diluted whole blood. (b) Sequence coverage generated by BUPID.
(a) Deconvoluted LTQ-CID MS2 spectrum obtained for the [M + 17H]17+ ion observed at m/z 932.632 that corresponds to the hemoglobin β chain variant exhibiting a −30-Da mass shift. (b) Sequence coverage generated by BUPID.
The NSD spectrum exhibited peaks that could be assigned as α chain fragments [b75 + 9H]9+ and [y61 + 8H]8+ at m/z 884.148 and 832.424, respectively, covering 136 of the 141 amino acids. While these pairs are not as complementary as of the pair selected for sequence analysis of TTR, they nevertheless cover close to 95% of the sequence. To some extent, the abundance of each of the chosen fragment ions can be increased by varying the fragmentation potential (by raising the skimmer voltage). To date, we have observed that implementation of the strategy using NSD to generate fragment ions which can be subsequently submitted to MS3 analysis is much more difficult for hemoglobins than for TTR.
This state of affairs is likely due to the fact that isolating and performing MS3
on some of the NSD-generated fragments is made more difficult when the peaks of interest have relatively low abundances and/or many other components are detected in the spectrum. This increases the possibility that NSD-generated fragment ions corresponding to the variant will appear at an m/z
values that are overlapped by interfering fragment ions. Furthermore, the presence of two proteins instead of one complicates the NSD fragment ion spectrum by simply increasing the number of fragments. Such overlap is not a problem when the intact protein charge states are chosen for MS2
analysis. The peaks due to wild-type and variants can be mass-selected together, without hindering the data interpretation, since all the sequence information obtained is common to wild-type and variant, except for those fragments that contain the amino acid substitution. In fact, it may not be necessary to apply, in all cases, the strategy based on the MS3
analysis of NSD-generated fragment ions that was found necessary and proven successful for TTR. Contrary to TTR, the intact protein MS/MS of some charge states of the intact α chain (particularly 14+ and 15+) show that it undergoes CID readily, yielding rich fragmentation and extensive sequence coverage. This is less true for the β chain where the “core” of the sequence was reported to be difficult to determine when CID was performed on the intact β chain [45
]. This report is consistent with our own observations. However, we have found that use of the NSD-generated β-y96
can resolve this issue. Hence, the best strategy in the case where the hemoglobin mass profile exhibits a β-chain variant, is to obtain the NSD spectrum and ascertain if the amino acid substitution is present within the y96
portion of the sequence. If so, the LTQ-CID MS3
spectrum of the variant y96
can be acquired. If not, MS/MS of intact protein from one of the charge states 14+ to 17+ can be used to gain sequence information.
On the basis of the results presented here, we can summarize the Top-down strategy for the analysis of hemoglobin variants as follows. First, a mass profile is obtained. If an α-chain variant is present, then LTQ-CID of the 15+ or 14+ charge state of the intact protein variant should be obtained. If a β-chain variant is found to be present, the NSD profile is acquired. If a variant β-chain y96 is detected, it can be analyzed by LTQ-CID MS3. Otherwise, NSD fragmentation and MS2 of the intact β-chain variant charge state (14+ to 17+) serves to characterize the variant.
The results obtained for a sickle cell variant (Hbβ Glu6→Val) illustrate the amount of information that can be extracted from the data. The mass profile of diluted whole blood known to contain a variant was obtained and an apparent β-chain variant with a −30-Da shift was observed (data not shown). The NSD spectrum did not exhibit a β-y96 variant peak; this result suggested that the amino acid substitution was present within positions 1–50. The β b-ion series revealed the presence of b-ion pairs (e.g., b13, b22 and b33) separated in each case by 30 Da. As noted earlier, the N-terminal region of the β-chain sequence may be determined by using MS2 of the intact protein charge states 14+ to 17+. In the MS/MS spectrum of the variant [M + 17H]17+ (), the −30-Da shift of the b-ions was observed down to b6, whereas the b4 mass corresponded to that of the wild-type sequence (). This data indicated that the amino acid substitution was at Pro5 or Glu6. In this case, the only possible amino acid substitution yielding a −30-Da shift is Glu6→Val6.
Table 2 List of neutral masses from the b-ion series of the deconvoluted LTQ-CID MS2 mass spectra of the [M + 17H]17+ of the wild type and Glu6Val hemoglobin β chain. This data indicated that the amino acid substitution was at Pro5 or Glu6. The only possible (more ...)
An added feature of the Top-down analysis of hemoglobins is the ability to characterize PTMs. The mass profile of a hemoglobin sample was recorded and the deconvoluted data indicated the presence of a minor component (10% abundance relative to the base peak series) corresponding to the molecular weight of the β chain + 305 Da. This mass shift is usually indicative of S
-glutathionylation at cysteine, a modification that is believed to be an indicator of oxidative stress [46
]. The low abundance [M + 19H]19+
peak at m/z
852.083 () (corresponding to glutathionylated β-chain) was subjected to LTQ-CID. The fragmentation information obtained () from this experiment was incomplete but was sufficient to place the modification on Cys93, and to exclude modification at Cys112. This method of locating the post-translational modification is considerably less cumbersome than the Bottom-up approach.
Figure 12 (a) Nanospray mass spectrum showing the expanded region containing the ion corresponding to [M + 19H]19+ of the S-glutathionylated hemoglobin β chain (m/z 852.08019+) (b) Sequence coverage generated by BUPID from the LTQ-CID MS2 of m/z 852.080 (more ...)