MS⁄ MS dissociation of molecular ions of an individual peptide can yield fragment masses that are indicative of its sequence.. In bot-tom-up identifications, the partial or full sequence
Trang 12 Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Austria
Introduction
The MS techniques of ESI [1] and MALDI [2] have
been available for only two decades, but they have
rev-olutionized the introduction of large, nonvolatile
mole-cules such as proteins into the mass spectrometer [3,4]
Here we discuss two general types of such MS
‘proteo-mics’ applications: (a) the identification of a protein
from among those predicted from the parent genome’s DNA; and (b) the structural characterization of a pro-tein, such as identifying and locating post-translational modifications (PTMs) or errors in the predicted sequence Currently, by far the most common method-ology for these in useful applications involves initial protein proteolysis, an approach that we have termed
‘bottom-up’ [5] The ‘top-down’ [5] approach described
Keywords
electron capture dissociation; MS; protein
characterization; protein identification;
post-translational modifications; top-down
proteomics
Correspondence
F W McLafferty, Baker Chemistry
Laboratory, Cornell University, Ithaca,
NY 14853, USA
Fax: +607 255 4137
E-mail: fwm5@cornell.edu
(Received 30 May 2007, revised 12 October
2007, accepted 17 October 2007)
doi:10.1111/j.1742-4658.2007.06147.x
For the characterization of protein sequences and post-translational modifi-cations by MS, the ‘top-down’ proteomics approach utilizes molecular and fragment ion mass data obtained by ionizing and dissociating a protein in the mass spectrometer This requires more complex instrumentation and methodology than the far more widely used ‘bottom-up’ approach, which instead uses such data of peptides from the protein’s digestion, but the top-down data are far more specific The ESI MS spectrum of a 14 protein mixture provides full separation of its molecular ions for MS⁄ MS dissocia-tion of the individual components False-positive rates for the identificadissocia-tion
of proteins are far lower with the top-down approach, and quantitation of multiply modified isomers is more efficient Bottom-up proteolysis destroys the information on the size of the protein and the connectivities of the pep-tide fragments, but it has no size limit for protein digestion In contrast, the top-down approach has a 500 residue, 50 kDa limitation for the extensive molecular ion dissociation required Basic studies indicate that this molecular ion intractability arises from greatly strengthened electro-static interactions, such as hydrogen bonding, in the gas-phase molecular ions This limit is now greatly extended by variable thermal and collisional activation just after electrospray (‘prefolding dissociation’) This process can cleave 287 inter-residue bonds in the termini of a 1314 residue (144 kDa) protein, specify previously unidentified disulfide bonds between eight of 27 cysteines in a 1714 residue (200 kDa) protein, and correct sequence predictions in two proteins, one of 2153 residues (229 kDa)
Abbreviations
BCA, bovine carbonic anhydrase; CAD, collisionally-activated dissociation; ECD, electron-capture dissociation; HAD, 3-hydroxyanthranilate-3,4-dioxygenase; IRMPD, infrared multiphoton dissociation; PFD, prefolding dissociation; PTM, post-translational modification;
PurL, formylglycinamide ribonucleotide amidotransferase.
Trang 2here directly introduces the proteins into the mass
spectrometer, providing far higher specificity at the
expense of far higher experimental requirements As
predicted in a prescient 2004 review [6], the top-down
method is being exploited increasingly in unique
appli-cations, with 18% of proteomics papers⁄ posters at the
2007 meeting of the American Society for Mass
Spec-trometry concerning this newer approach
Although ESI spectra of proteins larger than
mega-daltons have been reported [7,8], the great majority
of ESI spectra measured are those of the small
(< 3 kDa) peptides produced by the bottom-up
prote-omics methodology [9–13] The sample is digested with
a protease such as trypsin to produce a mixture of
small peptides from each protein, and is applicable to
even a complex mixture of proteins (e.g the ‘shotgun
approach’) [10] A common next step is the separation
of the total mixture into fractions by HPLC, followed
by their on-line introduction into the mass
spectrome-ter to yield ESI spectra showing molecular ions, and
thus molecular mass values, of the peptides MS⁄ MS
dissociation of molecular ions of an individual peptide
can yield fragment masses that are indicative of its
sequence These results can then be matched against
the molecular mass and MS⁄ MS peptide masses
expected for the individual proteins predicted from the
parent genome’s DNA In contrast [14], the ‘top-down’
methodology [5,6,14–22] can directly subject a mixture
of proteins, even of > 10 components, to ESI to yield
a spectrum of their molecular ions that indicates the
molecular mass values of individual proteins MS⁄ MS
of the mass-selected ions of a protein then provides
fragment mass values for its structural
characteriza-tion
In general, the bottom-up method is widely accepted
for the routine identification of proteins in complex
mixtures Usually, the identification of the gene that
encodes the protein is more important than full
struc-tural characterization of the protein Its quantitative
analysis by the bottom-up method under normal and
abnormal conditions can then provide a direct
indica-tion of the upregulaindica-tion or downregulaindica-tion of the
gene If, however, more extensive or specific data are
needed, such as on polymorphisms or PTMs, the
com-plementary top-down approach can often provide
these in a very straightforward manner This review
also discusses alleviation of a serious previous
prob-lem: top-down molecular ion dissociations have given
few product ions for proteins > 50 kDa The far
higher masses measured with the top-down approach
require correspondingly higher MS resolving power,
so the instrument of choice has been the expensive
Fourier transform mass spectrometer (FT MS)
[3,5,23,24] FT MS has the added advantage that it can give MS⁄ MS spectra by electron-capture dissocia-tion (ECD) [25–27], which provides far more fragment ion information than either collisionally-activated dissociation (CAD) [28] or infrared multiphoton disso-ciation (IRMPD) [29] However, ECD’s descendant, electron-transfer dissociation [30], works well with less expensive MS instruments, and can be applied to pep-tides and smaller proteins [31] with versatile ion–ion reactions [32] Of special promise for routine top-down applications is the recently developed Orbitrap mass spectrometer, which has resolution and mass accuracy capabilities approaching those of FT MS, with very promising cost advantages [33] ECD and electron-transfer dissociation are less sensitive than CAD or IRMPD, in part because they produce far more product ions
Identification
To date, by far the largest use of MS proteomics has been to identify unknown proteins, usually by match-ing mass values against those from a list of sequences predicted from the precursor DNA The quantities of these proteins that are expressed can differ by many orders of magnitude, so that a specific problem often requires preconcentration⁄ separation (e.g LC) In bot-tom-up identifications, the partial or full sequence of
an individual peptide is predicted from its molecular mass and MS⁄ MS mass values, with the number and uniqueness of these values determining the peptide identification accuracy The matching of multiple pep-tide sequences with that of a predicted protein increases the bottom-up identification accuracy, although it is possible that the same peptide data could also match those of another protein in the mixture (identified peptides that do not match a predicted pro-tein are typically ignored) Several bottom-up approaches achieve 1% identification accuracy in routine applications [9–13] Sensitivity, automation and throughput can also be of vital importance, but these depend on the combination of separation methods,
MS instrumentation, and computation employed Top-down MS⁄ MS of the selected molecular ion mass representing a specific protein produces far more fragments that have much higher masses, and are thus more unique, and the more expensive FT MS instru-mentation used with the top-down approach also pro-vides much higher mass accuracy [6,18,23,34] Furthermore, these fragment mass values originate from the same molecular ions, so they must all be characteristic of that protein’s sequence and molecular mass value Thus, top-down data can give an accuracy
Trang 3of identification that is orders of magnitude higher
[6,23,35] For example, Begley and co-workers [21]
iso-lated an enzyme YjbV involved in the B subtilis
thia-mine biosynthesis pathway for which 1D SDS-PAGE
analysis indicated an approximate mass of 3 kDa
(Fig 1) The top-down ESI⁄ FT MS spectrum of this
protein with nozzle-skimmer CAD dissociation (Fig 1)
confirmed the YjbV sequence and demonstrated the
absence of any post translational modifications Not
only does the measured molecular mass value of
31 407.1 agree with the predicted mass from the DNA
sequence at 31 406.9 Da, within the limits of
experi-mental accuracy, but also there are 23 top-down
frag-ment mass values that agree with those expected from
single backbone cleavages (Fig 1) Thus, each
frag-ment contains either the N-terminus or C-terminus,
providing extensive confirmatory sequence information
(see below) for this SDS⁄ PAGE-purified protein
For protein identifications in complex mixtures, a
dramatic advantage of the top-down approach is
that a final separation stage can be done in the
FT MS instrument For example, after rough
separa-tion of the proteins from Arabidopsis thaliana, the
stromal protein fraction was introduced directly by
ESI into the FT MS instrument to yield an ESI
mass spectrum in which the molecular ions from 14
different proteins can be distinguished (Fig 2) [20]
Figure 3 shows a protein’s molecular ion isotopic
cluster that yielded a measured molecular mass of
20 211.3 Da An obvious identification was the
DNA-predicted protein At1g06680, whose molecular mass is
20211.9 Da As a convincing confirmation, the CAD
MS⁄ MS spectrum of this isolated ion cluster included eight peaks of 8246–9308 Da whose mass differences matched those expected in the predicted protein for the sequence A-V-X4-F-G-G-(S + E) (Fig 3) [20] Extending this to mixtures of large proteins (see below), nozzle-skimmer dissociation spectra of 1 : 1,
2 : 1 and 3 : 1 mixtures of 144 and 116 kDa proteins showed the corresponding molecular ions and, for each, 11–17 different mass values of 1–10 kDa that represented their b or y fragment ions with a standard deviation of 5 p.p.m [36]
ECD The development of ECD [25] has made possible a dramatic increase in the proportion of inter-residue backbone bonds that can be cleaved in molecular ions The high-energy ( 5 eV) recombination of an electron with the multiply protonated ion makes differences in bond dissociation energies much less important and leads to much more indiscriminate protein backbone cleavages For example, 250 of the 258 inter-residue bonds could be cleaved (as assigned by the terminus-containing ions c, z., a., b and y) in bovine carbonic anhydrase (BCA) molecular ions in 25 ECD⁄ CAD spectra [19], with 183 bonds being cleaved in a single
‘plasma ECD’ spectrum (Fig 4) [26] Obviously, this amount of mass spectral information makes possible even higher identification reliabilities, and also extensive
de novosequencing and structural characterization
Fig 1 Left: 1D SDS ⁄ PAGE chromatograms
of ThiD from E coli and of unknown YjbV from B subtilis Right, above: ESI spectrum
of YjbV, molecular ion isotopic peaks Right, below: nozzle-skimmer dissociation spectral data, YjbV fragment peaks The ‘ ) 20’ after the molecular mass value signifies that the main component ion of the most abundant isotopic peak contains 20 13 C atoms and has this mass value.
Trang 4The high specificity of the top-down approach for
pro-tein structural characterization is due to the extensive
molecular connectivity information that it provides;
this is not destroyed by proteolysis The peptides from
proteolysis usually represent substantially less than
100% coverage of the protein sequence, so that even when their mass information is consistent with a previ-ously identified protein, the sample protein could have missing or extra parts In the top-down approach, an incorrect molecular mass value directly indicates the presence of PTMs and⁄ or an incorrect sequence In another ESI mass spectrum of proteins isolated from
Fig 2 ESI mass spectrum of the isolated
stromal proteins from A thaliana with their
measured molecular mass values [20].
Fig 3 ESI mass spectrum of the isolated chloroplast proteins from A thaliana (top) The 20 211.3 Da 19+ ions (< 10% abundance) were subjected to top-down MS⁄ MS to yield the CAD spectrum (bottom), which is consistent with the predicted sequence of At1g06680, molec-ular mass 20 211.9 Da.
Trang 5A thaliana [20], molecular ions representing a 5%
component gave a molecular mass of 16 309.7 Da, but
this matched none of the DNA-predicted proteins
MS⁄ MS of these ions gave the C-terminal sequence of
Fig 5 These and all other peaks of that spectrum did
match those expected for the predicted protein
At4g21280, although its molecular mass of
16 123.4 Da is lower than that found by 186 Da
Dissociation of the 16 121.8 Da fragment peak
(MS⁄ MS ⁄ MS, Fig 5) showed a fragment ion resulting
from an initial loss of 186.0 Da, followed by cleavages
corresponding to the N-terminal sequence of the
pre-dicted protein; the cleavage loss of the signal peptide
left two more amino acids on the protein than
pre-dicted Even if the bottom-up approach did provide
mass data on a peptide containing these amino acids,
these data would have been ignored in most protocols
However, even measuring a molecular mass value
that is the same as that predicted is not a guarantee
that the predicted sequence is correct In an early
(1993) example of top-down identification [23], our
measured molecular mass value, 29 024.2 Da, of BCA
matched well the value that was calculated,
29 024.7 Da, from the published sequence
Further-more, MS⁄ MS (nozzle-skimmer CAD) of the
molecu-lar ions gave 21 terminal fragment ions that were also
consistent with the published sequence However, our
2003 plasma ECD spectrum of BCA (Fig 4; 183
cleavage sites) gave 512 mass values [26], of which 45
were in error by ) 1 Da; these values all represented
cleavages in the region of residues 10–31 This is
strong evidence that the residue reported as Asp10
should be Asn10, and Asn31 should be Asp31
(Asp CO-OH, Asn CO-NH2, Dm ¼ –1 Da; note that
these changes do not affect the molecular mass value
of the protein) Detecting this error in the usual bot-tom-up approach would be difficult, as peptides that incorporate residues 10 or 31 would not match a pre-dicted sequence and so would be ignored Worse yet,
in our 1999 top-down study of BCA [5], + 1.00 Da and + 0.99 Da errors found for peptides Phe19– Asp33 and Asp18–Lys35 were termed ‘unexpected (and unexplained) anomalies’ Obviously, the precision
of locating such sequence errors or PTMs is depen-dent on obtaining fragment ion masses representing nearby dissociations on either side of the error; in the unusual Fig 4 case of nearby offsetting errors, having multiple ions representing cleavages between almost all neighboring residues made it clear that these were not ‘anomalies’
Post-translational modifications are the most com-mon challenge for the structural characterization of proteins Special bottom-up techniques have been developed for specific PTMs, e.g affinity separation of the protein digest to concentrate all glycosylated or all phosphorylated peptides for MS⁄ MS For a sample containing proteins modified on different sites, the bot-tom-up approach cannot characterize individual pro-teins In contrast, the top-down approach can select molecular ions with a molecular mass value cor-responding to, for example, a single substitution;
MS⁄ MS will then show the substituent positions of dif-ferent isomers A problem for MS⁄ MS of either the peptides for the bottom-up approach or of the proteins for the top-down approach is that backbone dissocia-tion techniques such as CAD or IRMPD can also cleave off side-chain substituents such as glycosylated, phosphorylated or sulfonated components, thus
Fig 4 A single plasma ECD spectrum of BCA whose 512 different m ⁄ z values define
183 of its 258 inter-residue cleavage sites [26] Of these m ⁄ z values, 45 are 1 Da higher than those predicted by the protein database sequence, and all represent cleav-ages between the proposed Asp10 and Asn31 This shows that these identifications are reversed, an error that does not affect the molecular mass value and a sequence consistent with those of sheep and human carbonic anhydrases.
Trang 6destroying information on their backbone location.
However, the energetic (‘nonergodic’) dissociation of
ECD is localized on the backbone, with little
accompa-nying cleavage of weaker side-chain modifications such
as glycosylated [37] and phosphorylated structures [38]
(and even of noncovalent bonding and conformational
tertiary protein structures; see below) Top-down ECD
and CAD of b-casein gave 126 out of the possible 208
backbone cleavages (Fig 6); the ECD cleavages not
only indicate the five phosphorylation sites without
loss of these side chains, but also that these cleavages
are so positioned that they would have specified
phos-phorylation if it had occurred at any of the other 21
possible sites (Ser, Thr, Tyr) of b-casein [38] Although
ECD requires the more expensive FT MS
instrumen-tation, it measures all product ions simultaneously,
which is of particular value for repeated quantitative
measurements, e.g variable phosphorylation of isolated
b-casein samples
Unexpected modifications are especially difficult for
classic and bottom-up methods, which must be selected
or tailored for the specific PTM In the biosynthesis of NAD, the enzyme 3-hydroxyanthranilate-3,4-dioxygen-ase (HAD) catalyzes the oxidative ring opening of 3-hydroxyanthranilate, which, with cyclization, forms a quinolinate [39] Excess quinolinate is implicated in neurological disorders such as stroke and Huntington’s disease, and 4-halohydroxyanthranilates have been found to be specific and potent HAD inhibitors To check for covalent modifications of the enzyme, the effect of the inhibitor on the molecular mass value of HAD was measured; instead of an adduct increase, or
no change, the value had unexpectedly decreased from
22 417.0 Da to 22 413.2 Da, a loss of 4 Da MS⁄ MS
of these molecular ions (Fig 7) cleaved 144 of the 193 inter-residue bonds (78 uniquely from ECD), confirm-ing almost completely the predicted sequence of the first 75 residues after eliminating the mistakenly pre-dicted N-terminal Met The fragment ions containing the C-terminus have the predicted mass values going back 10 residues to Cys183, but after Cys180 they are all 2 Da lower than predicted until Cys149 and
Fig 5 Partial CAD spectrum (top) of the
16+ ions of molecular mass 16 309.7 Da
(5% abundance) from ESI of the thylakoid
peripheral proteins isolated from A thaliana.
This spectrum matched the masses
pre-dicted for the C-terminus of the protein
At4G21280, molecular mass 16 123.4 Da.
A partial CAD spectrum (bottom) of the
16 121.8 Da 15+ fragment ions
(MS⁄ MS ⁄ MS) matched that protein’s
N-ter-minus plus two signal peptide amino acids
whose mass corresponds to the 186 Da
dis-crepancy in the protein molecular mass
value.
Trang 7Cys146, after which they are low by 4 Da, the
decrease of the molecular mass value The most
proba-ble reason for a 2 Da decrease is the formation of an
S–S bond; although this was totally unexpected and
unprecedented, the top-down approach efficiently gave
a specific characterization of the inhibitor mechanism
[39] Even if two S–S bonds had been suspected,
identi-fying for each their two specific cysteines cut of the
10 possible for the five cysteines (including Cys127),
would be difficult by classic or bottom-up methods
Deamidation of Asn or Gln in proteins has
impor-tant effects on enzyme activity and folding, and has
even been proposed as a biological clock [40] How-ever, changing –CO-NH2to –CO-OH only produces a mass increase of 1 Da; as in Fig 4, this makes the ability of FT MS to resolve protein ion isotopic peaks
of critical importance for such a mass shift determina-tion The most abundant of the 13+ molecular ions of reduced RNase A before deamidation (Fig 8A) shows
Fig 7 ECD, CAD and IRMPD spectral data of HAD treated with
inhibitor [22] C-terminal fragment ions 1–4 Da below the mass
values predicted for untreated HAD clearly indicate the unexpected
S–S bonds Cys146 to Cys149 and Cys183 to Cys186.
Fig 6 Inter-residue backbone fragmentations from the ECD
spect-rum of b-casein’s three variants, molecular masses 24 008.2 Da,
23 968.2 Da, and 24 077.2 Da [38] These fragmentations are
con-sistent with the known phosphorylations at Ser15, Ser17, Ser18,
Ser19, and Ser35 These fragmentations would also specifically
indicate any phosphorylation that occurred at the other 21 possible
Ser, Thr and Tyr sites.
Fig 8 Molecular ion isotopic clusters from ESI of the product mix-tures from deamidation of RNase A over increasing time periods Deamidation of any one of the 17 Asn and Gln sites of RNase A produces a 1 Da increase in the mass, –CO-NH 2 fi –CO-OH, of the molecular ions of that product The observed isotopic abun-dances give calculated best fits for the average increases of 0.0, 1.0, 1.8, 3.7 and 4.4 Da, respectively, in the masses of the prod-ucts [40].
Trang 8a mass of 13 689.3 Da versus the calculated value
13 689.3 Da The circles represent the calculated
abun-dance distribution for the isotopic peaks whose
maxi-mum peak contains mainly 13C8, whereas the squares
represent the distribution 1 Da higher To determine
the mass increase with increasing time of deamidation
(pH 9.6), the best fit of calculated intensity values
(squares) was determined (Fig 9B–E) The
correspond-ing mass increase values in the ECD and CAD
frag-ment ions were determined similarly and are plotted
for the four product samples in Fig 9 as mass
increases (decreases) for the N-terminal
(C-terminal)-containing fragment ions Thus, for the + 1.0 Da
sam-ple (Fig 8B), the N-terminal fragment ions show little
increase in mass with increasing size until Asn67, with
this increase of 1.0 Da staying constant for larger
N-terminal ions and with the C-terminal ions showing
the complementary decrease This demonstrates
directly that Asn67, the only deamidation site found
previously, is indeed deamidated before any other
resi-due In a similar fashion, the samples with 1.8, 3.7 and
4.4 Da increases show that Asn71 and Asn94 are
nearly equally reactive as the next sites, followed by
Asn34 and then Gln74 [40] Other examples show the utility of top-down MS⁄ MS for such kinetic studies [17,22,41]
Top-down quantitative analysis Measuring the differences in protein expression levels that result from disease states, environment, etc is critically important in many biomedical investiga-tions The protein quantities in cases of normal and perturbed expression are compared accurately by iso-topically labeling the proteins from one and compar-ing in their mixture the correspondcompar-ing peaks of their respective peptides, usually differing by three or more mass units [9–12] The kinetic deamidation study above (Fig 9), in a similar fashion, compares the quantities of proteins differing in the position of deamidation (only a + l Da change) with the multi-ple MS⁄ MS spectral peaks, providing multiple mea-surements of the quantities The top-down approach should be the method of choice for quantitation of position isomers of proteins containing multiple mod-ifications [42]
Fig 9 ECD spectral data from the RNase A deamidation samples of Fig 8 Deamidation at an individual residue of a specific product causes
a 1 Da increase in any fragment ion containing that residue The average mass gain of N-terminal and C-terminal fragment ions are plotted
as positive and negative, respectively, mass increases, with the molecular ion mass increases of Fig 8 designated on the right ordinate [40].
Trang 9dissociation used for the bottom-up approach is far
more generally applicable than the gas-phase MS⁄ MS
dissociation methods used with protein molecular ions
for the top-down approach With increasing protein
size, the hydrophilic (e.g hydrogen bonds) and
hydro-phobic tertiary bonding becomes more complex and
stabilizing Such native conformer structures of
pro-teins in solution are easily destroyed by various
reac-tive agents, but top-down dissociation methods for
gaseous protein ions, such as CAD and IRMPD, are
unimolecular, and so require the use of increasing
amounts of energy for the dissociation of increasingly
large protein ions Basic studies over the past 15 years
have shown fundamental differences in protein
confor-mations in solution versus the gas phase, with H⁄ D
exchange identifying reactive regions of the
conforma-tion [43–45], ion mobility measuring conformaconforma-tional
cross-sections [46,47], ECD identifying regions of
ter-tiary noncovalent bonding, as these are preserved when
backbone bonds are cleaved [48,49], and infrared
photodissociation spectroscopy characterizing
func-tional group environments [44,50] For example,
charge sites, such as the protonated side chains of
basic residues, in solution are solvated out into the
aqueous phase, while in the gas phase they are instead
solvated onto the protein backbone, with this
appar-ently favored if the backbone is in an a-helical
struc-ture [44–50]
ECD itself causes negligible cleavage of this tertiary
structure However, its noncovalent bonds have
sub-stantially lower bond dissociation energies, in general,
so that limited activation by earlier or concurrent
this tertiary structure has become so strong and exten-sive that conventional activation by CAD or IRMPD gives few or no backbone cleavages, making the top-down approach ineffective [51]
A possible solution to this problem was indicated by the study of conformational changes occurring during solvent evaporation immediately after electrospray introduction into the FT mass spectrometer [52] Solu-tion protein conformaSolu-tions are actually unfolded dur-ing electrospray; use of native ECD [53] showed that ECD could occur without externally added electrons when electrosprayed native cytochrome c unfolded in the inlet capillary, exposing basic residues that attracted electrons and caused ECD Solvent removal reduces or destroys hydrophobic bonding Further-more, in solution, water molecules solvate the protein’s protonated side chains; on solvent removal, these are immediately available for new hydrogen bonding Thus, supplying thermal and collisional energy during solvent evaporation can slow the new folding stabiliza-tion of the protein ions, while also providing sufficient excitation to effect cleavage before the gaseous confor-mation becomes too stable [52]
This new technique of prefolding dissociation (PFD) has now been successfully applied to 116, 144, 200 and
229 kDa proteins [36], using a 6 Tesla FT MS instru-ment [15–17] ESI of formylglycinamide ribonucleotide amidotransferase (PurL) (1315 residues), whose reported sequence [54] corresponds to a molecular mass of 143 635 Da, gave the Fig 10 spectrum indicating a molecular mass of 143 500 ± 23 Da Our nozzle-skimmer dissociation system can vary the ion
Fig 10 ESI mass spectrum of PurL Isotopic peaks are not resolved; deconvolution yields a molecular mass of 143 500 ± 23 Da [36].
Trang 10accelerating voltage for CAD both in the 1 Torr
pressure region before the skimmer (Vpre) and in the
10)3Torr region after the skimmer (Vpost) In
gen-eral, Vpre produces many low-energy collisions to
cleave noncovalent bonds, whereas Vpost produces
fewer collisions with energies approaching the
acceler-ating voltage to cleave backbone bonds Different
combinations of Vpre, Vpost and capillary temperature
values in 11 PFD spectra gave 173 different
inter-resi-due backbone cleavages (Fig 11) In a serendipitous
discovery, additives to the ESI solution such as
ammo-nium tartrate increased the number of cleavages by
50%, with a total of 21 spectra showing 287
differ-ent cleavages (Fig 11) [36] These are only between the
first 240 residues from each end, so that here they
provide extensive ( 60%) sequence coverage For
example, these data clearly show that the predicted
N-terminal Met is not present; this changes the
pre-dicted molecular mass value to 143 504 Da, in good
agreement with that found of 143 500 ± 23 Da
How-ever, no information has been obtained from the
central 900 residues; we picture this gaseous protein
conformation as a ‘ball of spaghetti’, for which the
energetic activation has denatured the free ends or has
prevented them from folding Possibly, the highly
ener-getic ECD in the capillary-skimmer region could effect
a few cleavages in the center of the protein to form
additional loose ends to be denatured out of the ball
of spaghetti
The ESI spectrum of the 200 kDa human
comple-ment C4 glycoprotein (of 1714 residues in three chains
connected by three S–S bonds) [55] had no molecular
ions Nearly complete deglycosylation (of predicted
molecular mass 186 437 Da) was indicated, as gentle
PFD gave fragment ions of 20 838 Da (b-185 of the
b-chain) and 165 746 ± 80 Da, with the total
186 584 ± 80 Da indicating < 0.1% remaining
glyco-sylation This was confirmed by stronger PFD, with
which 87 fragment ions were found to correspond to
different cleavages of the deglycosylated protein This contains 27 Cys residues, but it was not known which are still in the –SH form or which form S–S bonds, and what are the connectivities for the latter As for HAD [39] above, the presence of an S–S bond in a ter-minal fragment ion causes the PFD fragment mass to
be 2 Da less than the sequence-predicted value, and fragment ions are usually not observed from cleavages between the Cys residues, as they are held together by the S–S bond With this, eight additional S–S bonds could be specified [36]
The largest protein examined, mycoserosic acid synthase, had a predicted [56] molecular mass of
229 067 da (2154 residues), whereas ESI gave
228 934 ± 60 Da Five PFD spectra designated 62 cleavages by omitting the predicted N-terminal Met, correcting the molecular mass value to 228 936 Da to agree with that measured Its ‘ball of spaghetti’ is more difficult to unravel; cleavages were limited to 134 and
182 residues from the N-terminus and C-terminus, respectively Very recently in collaboration with
M Boyne and N Kelleher, (University of Illinois, Urbana, IL) PFD has also been implemented on an 8.4 Tesla FT MS instrument, despite its substantially different ion entrance system, which includes an ion funnel and octupole for ion storage
Conclusions The top-down and bottom-up proteomics approaches are obviously complementary The identification of proteins from among those predicted by the DNA sequence still has by far the largest sample demands
In most cases, the bottom-up approach, requiring less sophisticated instrumentation and expertise, should be tried first for qualitative identification, although increas-ing demands for more accurate quantitation provide a promising area for the top-down approach [36,57,58] Reliability of identification can be far superior with the
Fig 11 PFD spectral data of PurL Inter-residue backbone fragmentations are indicated by: N-terminal-containing b fragment ions (left, above line); C-terminal-containing y ions (right, above line); and secondary fragment ions (below line) Top line: 173 different fragmentations from
11 spectra using various values of capillary temperature and preskimmer and postskimmer accelerating voltages Bottom: 287 in total, including 10 additional spectra with ammonium tartrate added to the ESI solution.