Ultraviolet photodissociation at 355 nm of fluorescently labeled oligosaccharides

Article abstract of DOI:10.1021/ac800315k

Ultraviolet photodissociation (UVPD) produces complementary fragmentation to collision-induced dissociation (CID) when implemented for activation of fluorescently labeled oligosaccharide and glycan ions. Reductive amination of oligosaccharides with fluorophore reagents results in efficient photon absorption at 355 nm, producing fragment ions from the nonreducing end that do not contain the appended fluorophore. In contrast to the fragment ions observed upon UVPD (A- and C-type ions), CID produces mainly reducing end fragments retaining the fluorophore (Y-type ions). UVPD affords better isomeric differentiation of both the lacto-N-fucopentaoses series and the lacto-N-difucohexaoses series, but in general, the combination of UVPD and CID offers the most diagnostic elucidation of complex branched oligosaccharides. Four fluorophores yielded similar MS/MS results; however, 6-aminoquinoline (6-AQ), 2-amino-9(10H)-acridone (AMAC) and 7-aminomethylcoumarin (AMC) afforded more efficient photon absorption and subsequent dissociation than 2-aminobenzamide (2-AB). UVPD also was useful for characterization of glycans released from ribonuclease B and derivatized with 6-AQ. Lastly, electron photodetachment dissociation of oligosaccharides derivatized with 7-amino-1,3-naphthalenedisulfonic acid (AGA) yielded unique cross-ring cleavages similar to those obtained by electron detachment dissociation.

Full text of DOI:10.1021/ac800315k

Anal. Chem. 2008, 80, 5186–5196
Ultraviolet Photodissociation at 355 nm of
Fluorescently Labeled Oligosaccharides
Jeffrey J. Wilson and Jennifer S. Brodbelt*
Department of Chemistry and Biochemistry, 1 University Station A5300, University of Texas at Austin,
Austin, Texas 78712
Ultraviolet photodissociation (UVPD) produces comple-
mentary fragmentation to collision-induced dissociation
(CID) when implemented for activation of fluorescently
labeled oligosaccharide and glycan ions. Reductive ami-
nation of oligosaccharides with fluorophore reagents
results in efficient photon absorption at 355 nm, produc-
ing fragment ions from the nonreducing end that do not
contain the appended fluorophore. In contrast to the
fragment ions observed upon UVPD (A- and C-type ions),
CID produces mainly reducing end fragments retaining
the fluorophore (Y-type ions). UVPD affords better iso-
meric differentiation of both the lacto-N-fucopentaoses
series and the lacto-N-difucohexaoses series, but in
general, the combination of UVPD and CID offers the most
diagnostic elucidation of complex branched oligosaccha-
rides. Four fluorophores yielded similar MS/MS results;
however, 6-aminoquinoline (6-AQ), 2-amino-9(10H)-ac-
ridone (AMAC) and 7-aminomethylcoumarin (AMC) af-
forded more efficient photon absorption and subsequent
dissociation than 2-aminobenzamide (2-AB). UVPD also
was useful for characterization of glycans released from
ribonuclease B and derivatized with 6-AQ. Lastly, electron
photodetachment dissociation of oligosaccharides deriva-
tized with 7-amino-1,3-naphthalenedisulfonic acid (AGA)
yielded unique cross-ring cleavages similar to those
obtained by electron detachment dissociation.
Glycans are particularly challenging to study due to their
structural diversity stemming from the various nonlinear branch-
ing structures as well as their array of intersaccharide linkages.
Moreover, the position of their attachment within the protein
structure as well as the type of glycosylation, N- or O-linked, is
essential for comprehensive mapping of glycoproteins. N-Glyco-
sylation solely occurs through the creation of an N-glycosidic bond
with Asn where the N-linked amino acid position sequence follows
Asn-X-Ser/Thr (where X cannot be Pro), whereas O-glycosylation
occurs via Ser or Thr residues in proteins. These modifications
can reside not only at multiple locations throughout the proteins
but also with a variety of different glycan forms attached to each
individual site. The rigorous identification of these complex glycan
structures typically requires several analytical techniques such
as MS, NMR, endo- and exoglycosidase digestions, liquid chro-
matography, and monosaccharide analysis used collectively for
complete characterization.11,12
Fluorescent labeling of oligosaccharides is frequently employed
for the enhancement of the detection sensitivity as well as improve-
ment of separation in HPLC and fluorophore-assisted capillary
electrophoresis (FACE) analysis.13,14 An assortment of fluorophores
have been evaluated with no universal reagent yet providing
unsurpassed results for all types of oligosaccharide analysis.14
Charged fluorophores are typically employed for FACE analysis in
which the charged character facilitates separation. On the other hand,
the hydrophilic nature of neutral and acidic oligosaccharides makes
their separation difficult by HPLC; therefore, hydrophobic fluoro-
phores are desired for improving separation characteristics.14 Ad-
ditionally, fluorophores differ in both their spectroscopic properties
and reaction yields, which further influences their suitability for
particular analytical objectives.14 The most common method for
appending a fluorophore to an oligosaccharide has been via reductive
amination through the reducing end sugar via a Schiff base inter-
mediate.15 These commonly used derivatization strategies also make
them a natural approach for implementation of ultraviolet photodis-
Glycomics has become a research field of great interest as it
provides a window into understanding an assortment of cellular
functions related to health and disease.1 Oligosaccharides as well
as glycans (oligosaccharides released from glycoproteins) par-
ticipate in a variety of cell processes such as cellular signaling
and differentiation, virus infection and replication, immune re-
sponse, and inflammation in addition to regulation of biochemical
pathways.2–9 Furthermore, glycosylation is now recognized as the
most common posttranslational modification of eukaryotic proteins
in which greater than 50% of these proteins can undergo glyco-
sylation.10
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* To whom correspondence should be addressed. E-mail: jbrodbelt@
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5186 Analytical Chemistry, Vol. 80, No. 13, July 1, 2008
10.1021/ac800315k CCC: $40.75 2008 American Chemical Society
Published on Web 05/28/2008

Scheme 1. Reductive Amination Reaction
Involving a Reducing End Sugar and Fluorescent
Label AMCa
been employed for the structural determination of oligosaccha-
rides, glycans, and glycopeptides. The latter photodissociation
(PD) methods have predominantly used in FTICR and quadrupole
ion trap (QIT) mass spectrometers. Photodissociation provides
several distinct advantages over traditional CID in QIT mass
spectrometers. Ion activation by photodissociation is independent
of the trapping rf voltage, thus allowing efficient retention of
fragment ions over a broad m/z range. Moreover, the nonresonant
nature of PD results in the activation of all ions, not just the
selected precursor, which causes informative secondary dissocia-
tion and bypasses the need for complex MSn scan functions. These
advantages of QITs have been exploited in the analysis of an array
of biomolecules ranging from oligosaccharides35,37 and DNA,39–43
to peptides and proteins.44–58
There have been several previous studies that utilized PD
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a The reaction forms a Schiff base intermediate, which under-
goes reduction to yield a stable linkage between the fluorophore
and oligosaccharide structure.
.
.
.
.
sociation (UVPD) mass spectrometry for structural characterization
of oligosaccharides in which the attached chromophores enhance
UV absorptivity for ion activation, as illustrated in the present study.
In general, tandem mass spectrometry has proven to be a
powerful tool for the structural analysis of biomolecules, especially
in proteomics and genomics,16–18 and also for the identification
of glycans.19,20 For example, collision-induced dissociation (CID),
the most widely used activation method, principally results in the
formation of diagnostic B-/Y- and C-/Z-type fragment ions for
unmodified oligosaccharides (using the Domon and Costello
nomenclature as illustrated in Scheme 2).21,22 Other activation
methods, including postsource decay,23,24 electron capture
dissociation,25–27 electron detachment dissociation (EDD),28,29
electron-transfer dissociation,30,31 infrared multiphoton dissociation
(IRMPD),32–36 and more recently UVPD37,38 at 157 nm, have all
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Analytical Chemistry, Vol. 80, No. 13, July 1, 2008 5187

Scheme 2. Oligosaccharide Fragmentation Nomenclature Adapted from Domon and Costello
Nomenclaturea 21,22
a The bottom scheme shows the -AMC* and -AMC′′ nomenclature used to represent the resulting 0,1Ax and 0,2Ax product ions,
respectively.
mass spectrometer.34 Xie et al. noted that the varying IRMPD
dissociation thresholds of alkali metal cationized oligosaccharides
enhanced isomer differentation.33 An IR wavelength-tunable free
electron laser for infrared experiments was used by Fukui et al.
to investigate the optimal dissociation wavelengths of sodium-
cationized oligosaccharides.36 Pikulski et al. reported that oli-
gosaccharides derivatized with an IR-chromogenic boronic acid
underwent efficient IRMPD and extensive secondary dissociation
in a QIT, producing fragment ions that result via cleavage from
only the nonreducing ends.35 In terms of UVPD of oligosaccha-
rides, Devakumar et al. reported two studies utilizing an excimer
laser at λ ) 157 nm.37,38 UVPD at 157 nm induced extensive cross-
ring cleavages, which provided key information about saccharide
linkage positions.
In this study, we report the merits and challenges associated
with the application of UVPD at 355 nm to fluorescently labeled
oligosaccharides in a QIT mass spectrometer. While CID produces
fragment ions retaining the fluorophore attached through the
reducing end sugar, UVPD provides complementary information
for these complex structures via the formation of fragment ions
without the fluorophore and instead emphasizing the nonreducing
end of the oligosaccharide. UVPD affords streamlined isomeric
differentiation of various oligosaccharide branching structures.
Electron photodetachment dissociation (EPD) experiments are
also conducted on oligosaccharides modified with an acidic
fluorophore reagent to induce radical type cleavages.
ics; 7-amino-4-methylcoumarin (AMC) from MP Biomedicals, Inc.
(Irvine, CA); and 2-amino-9(10H-)acridone (AMAC) and 7-ami-
nonaphthalene-1,3-disulfonic acid (AGA) from AnaSpec (San Jose,
CA). These fluorescent reagents are displayed in Figure 1. All
oligosaccharides were purchased from V-Laboratories (Covington,
LA) except lacto-N-difucohexaose (LNDFH)-Ib, which was pur-
chased from Sigma Aldrich. The lacto-N-fucopentaose (LNFP) and
LNDFH oligosaccharide series are displayed in Figure 2. Glycerol
free peptide N-glycosidase F (PNGase F) was purchased from
New England Biolabs (Ipswich, MA).
Deglycosylation. Enzymatic treatment of glycoproteins was
conducted following the manufacturer’s protocol. In short, 20 µg of
a glycoprotein solution was diluted into 10 µL of 0.5% sodium dodecyl
sulfate and 0.04 M dithiothreitol solution and then heated to 100 °C
for 10 min for denaturing. Next the solution was compensated to
create a final concentration of 1% Nonidet P-40 and 50 mM Na2PO4
prior to the addition of 5 units of PNGase F. This solution was
incubated at 37 °C for 2 h prior to subjecting the released glycans in
this solution to reductive amination derivatization.
Derivatization. Reductive amination of oligosaccharides and
glycans were conducted using a reductive amination procedure
previously described by Prime et al.15 A 10 µL oligosaccharide
solution (LNFP series and LNDFH series) at a concentration of
500 µM or ∼10 µg of released glycans were mixed with a 10 µL
solution of the amine-containing fluorophore (∼0.5 M) in 7:3
DMSO/HOAc solution, which was heated for 1 h at 65 °C. Twenty
microliters of 1 M NaCNBH3 was added to this solution and heated
for another hour at 65 °C. Derivatized oligosaccharides and
glycans were desalted and removed from excess fluorophore by
porous graphitic carbon Carbograph Extract-Clean SPE columns
EXPERIMENTAL SECTION
Reagents. All chemicals were purchased from Sigma Aldrich
(St. Louis, MO) except the following: 6-aminoquinoline (6-AQ)
and 2-aminobenzamide (2-AB) were obtained from Acros Organ-
5188 Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

control unit for triggering the laser. The laser was operated at 10
Hz with full energy pulses at (∼60 mJ/ pulse) with the number
of pulses reported unless otherwise noted.
RESULTS AND DISCUSSION
This comparative study of CID and UVPD methods applied to
fluorescently labeled oligosaccharides was conducted to evaluate the
diagnostic value of the resulting MS/MS spectra for determination
of the branching structures. A previous study by our group reported
the utility of 355 nm UVPD applied to chromophore-derivatized
peptides in which several distinct advantages over traditional CID
methods were highlighted.55 Among these advantages, the secondary
dissociation of chromophore-containing fragment ions upon UV
irradiation was particularly valuable for providing a greater array of
diagnostic sequence ions. Oligosaccharides, much like other biopoly-
mers, do not readily absorb photons over large regions of the
UV-visible light spectrum and therefore must be modified to
undergo efficient photodissociation at 355 nm. However, unlike
proteins and nucleic acids, oligosaccharides are far more difficult to
analyze by conventional spectroscopic methods, a factor that has led
to the frequent utilization of derivatization strategies to improve
detection limits.14 The incorporation of amine-containing fluoro-
phores, one common approach, also makes the oligosaccharides
amenable to UVPD mass spectrometry. Fluorophores appended to
oligosaccharides by reductive amination include 2-AB, AMAC, AMC,
6-AQ, and AGA (see Figure 1), all of which promote UV absorption
at λex ∼ 355 nm. These amine-containing reagents selectively react
with the anomeric carbon located on the reducing end of the
oligosaccharide, which restricts the derivatization of these analytes
to one specific site at the core of the structure. The reaction proceeds
through a Schiff base intermediate, which is then reduced to form a
stable linkage between the reducing end sugar and the appended
fluorophore as depicted in Scheme 1.
MS/MS Characterization of the LNFP Oligosaccharide
Series. The elucidation of branching structures of isomeric
oligosaccharides has been accomplished previously by tandem
mass spectrometry. For example, CID of sodium-cationized
oligosaccharides typically yields fragment ions containing the
reducing end sugar. As an example, the CID mass spectra of
sodium-cationized LNFP-I, -II, -III, and -V are shown as
Supporting Information (Figure S1) for benchmark purposes.
The chemical and symbolic structures of these oligosaccharides
are shown in Figure 2 for comparison. After derivatization with
the fluorophore 6-AQ, the LNFP oligosaccharides also produce
reducing end fragments upon CID (Figure 3). All mass spectra
in this study are labeled with arrows to indicate possible sugar
losses (which do not necessarily imply specific fragmentation
pathways) as well as accepted oligosaccharide fragmentation
nomenclature (A, B, C, Y) ions.21 In Figure 3, partial dif-
ferentiation of the LNFP-I, -II, and -III isomers from the LNFP-V
isomer is possible based on the presence of the B2 fragment
ion in the CID mass spectra of the former and the 0,3A4 ion in
the CID mass spectrum of the latter. The diagnostic fragment
ions that specifically allow isomer differentiation are labeled
with an inverted triangle (3) symbol in the mass spectra for
easy recognition throughout this study. Unfortunately, these
CID mass spectra are not sufficiently unique to allow successful
differentiation of the first three LNFP isomers.
Figure 1. Structures of the fluorescent reagents for reductive
amination of oligosaccharides. In parentheses next to the name is
the chemical abbreviation. The nominal mass addition to the oli-
gosaccharide is included in brackets.
(Alltech Associates, Inc., Deerfield, IL) following the protocols
described by Packer et al.59
Mass Spectrometry. A ThermoFinnigan LCQ Deca XP (San
Jose, CA) modified for UVPD, was employed for all mass
spectrometry experiments as further described in the next
section.55 Analyte solutions were diluted to a concentration of 10
µM in a 50/50 ACN/H2O (v/v) solvent mixture for ESI-MS
analysis. These solutions were infused into the mass spectrometer
at a flow rate of 3-5 µL/min with a Harvard Apparatus PHD 2000
syringe pump (Holliston, MA). Ion activation by CID was
conducted at the default qz values of 0.25 and 30 ms. The CID
and UVPD MS/MS spectra were characterized with the aid of
the web application GlycoFragment.60
Ultraviolet Photodissociation. UVPD experiments were
performed on an LCQ Deca instrument previously described in
full detail.55 In brief, this mass spectrometer was equipped with
an unfocused Quanta-Ray GCR-11 Nd:YAG laser with a HG-2
harmonics generator from Spectra-Physics (Mountain View, CA)
for the creation of 355 nm photons. A CF viewport was incorpo-
rated into the top vacuum flange mount for transmission of the
UV photons through an antireflective quartz window from CVI
laser (Albuquerque, NM). The Nd:YAG laser was gated by a TTL
signal output from the instrument, which was controlled by the
instrument software. This TTL signal was increased to 15 V
through an operational amplifier circuit integrated into the laser
(59) Packer, N. H.; Lawson, M. A.; Jardine, D. R.; Redmond, J. W. Glycoconjugate
J. 1998, 15, 737–747
(60) Lohmann, K. K.; Von Der Lieth, C.-W. Nucleic Acids Res. 2004, 32, W261-
W266
.
.
Analytical Chemistry, Vol. 80, No. 13, July 1, 2008 5189

Figure 2. Chemical and symbolic structures of the LNFP oligosaccharide series and the LNDFH oligosaccharide series.
The corresponding UVPD mass spectra for the same four
isomeric fluorophore-derivatized, sodium-cationized oligosac-
charides display vastly different information from the CID mass
spectra, as shown in Figure 4. The most notable difference is
that the detected fragment ions stem from the nonreducing
end of the oligosaccharide, which primarily yields A- and C-type
ions, as opposed to the dominant Y-type ions produced upon
CID. Another common feature of the UVPD spectra is the loss
of the fluorophore group in conjunction with part of the
reducing sugar, and these characteristic losses are labeled as
-6-AQ* and -6-AQ′′, corresponding to formation of 0,1A- and
0,2A-type fragment ions, respectively. These types of fragments
are illustrated in the bottom structure of Scheme 2. The
complementary loss of the remaining reducing end monomeric
sugar moiety via cleavage of the glycosidic bond is labeled as
-Glc*. In general, the UVPD mass spectra show more unique
fragments than the CID mass spectra, thus enhancing isomer
differentiation of the LNFP oligosaccharides. For example, the
additional loss of the fructose group (-Fuc) from the 0,2A5
fragment is observed only for the LNFP-I isomer in the UVPD
mass spectrum (Figure 4A). Additionally, new fragment ions
such as the C2 and C2/Y3R ions can be utilized for differentiation
of LNFP-II and -III from the LNFP-V isomers. However, the
LNFP-II and -III isomers remain indistinguishable from one
another even upon UVPD, presumably due to the similarities
in their structures, differing only by their Fuc linkage to GlcNAc
(R1-3 vs R1-4), which requires highly specific cross-ring
cleavages at this position for identification. Similar results as
those highlighted in Figures 3 and 4 are obtained when the
other fluorophores are attached to these four oligosaccharides.
These results are promising in that CID and UVPD provide
complementary MS/MS information by affording sequence ions
5190 Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

Figure 3. ESI-MS/MS spectra of (A) [LNFP-I + Na + 6-AQ]+, (B) [LNFP-II + Na + 6-AQ]+, (C) [LNFP-III + Na + 6-AQ]+, and (D) [LNFP-V
+ Na + 6-AQ]+ by CID. The triangle symbol (3) represents unique fragments useful in isomeric differentiation. Loss of the fluorophore reagent
and reducing sugar glucose is labeled -6-AQ+Glc. A star symbol (f) is used to signify the precursor ion. The magnification scale bar applies
to all spectra over the indicated mass range.
Figure 4. ESI-MS/MS spectra of (A) [LNFP-I + Na + 6-AQ]+, (B) [LNFP-II + Na + 6-AQ]+, (C) [LNFP-III + Na + 6-AQ]+, and (D) [LNFP-V
+ Na + 6-AQ]+ by UVPD using 20 pulses at 60 mJ/pulse for all four spectra. The triangle symbol (3) represents unique fragments useful in
isomeric differentiation. Loss of the fluorophore reagent by cross-ring cleavage of the reducing sugar is labeled -6-AQ* and -6-AQ′′, representing
formation of 0,1A- and 0,2A-type fragment ions, respectively. Loss of the fluorophore reagent and reducing sugar glucose is labeled -6-AQ+Glc.
A star symbol (f) is used to signify the precursor ion. The magnification scale bar applies to all spectra over the indicated mass range.
from opposite ends of the oligosaccharides, a particularly
desirableoutcomeforanalysisofcomplex, nonlinearbiopolymers.
MS/MS Characterization of the LNDFH Oligosaccharide
Series. The same features noted in the UVPD and CID mass
spectra of the fluorescently labeled LNFP series are relevant for
the differentiation of a more complex series of oligosaccharides,
namely the LNDFH series. The CID mass spectra of the sodium-
cationized LNDFH isomers are displayed in the Supporting
Information section (Figure S2). For comparative purposes, a
second fluorophore reagent, AMC, was used instead of 6-AQ to
Analytical Chemistry, Vol. 80, No. 13, July 1, 2008 5191

Figure 5. ESI-MS/MS spectra of (A) [LNDFH-Ia + Na + AMC]+, (B) [LNDFH-Ib + Na + AMC]+, and (C) [LNDFH-II + Na + AMC]+ by CID.
The triangle symbol (3) represents unique fragments useful in isomeric differentiation. Loss of the fluorophore reagent by cross-ring cleavage
of the reducing sugar is labeled -AMC′′, yielding the 0,2A-type fragment ion. A star symbol (f) is used to signify the precursor ion.
derivatize this series of compounds. The resulting CID and UVPD
mass spectra for the sodium-cationized fluorophore-labeled oli-
gosaccharides are shown in Figures 5 and 6, respectively. The
chemical and symbolic structures of these LNDFH oligosaccha-
rides are shown in Figure 2. The CID mass spectra obtained for
the LNDFH-Ia and LNDFH-Ib complexes, Figure 5A and B,
respectively, reveal similar fragmentation patterns, which is typical
for isomers differing only in their Fuc-GlcNAc linkage, in this case
R1-3 versus R1-4. The CID mass spectrum for the third isomer,
LNDFH-II (Figure 5C), reveals one new fragment ion attributed
to the loss of the fluorophore from the reducing sugar (0,2A4/
Y3R”/1ꢀ, labeled 3) that could differentiate this isomer; however,
the abundance of this single unique fragment ion is low, thus
limiting the confidence in identifying this oligosaccharide.
The corresponding UVPD mass spectra for these AMC-labeled
LNFDH oligosaccharides display notably different fragmentation
patterns as shown in Figure 6. Again the major fragment ions do
not contain the fluorophore and stem from the nonreducing end
of the oligosaccharide, yielding A- and C-type ions as opposed to
the Y-type ions created by CID. Cross-ring cleavages of the
reducing sugar containing the fluorophore that occur for the
LNDFH isomers result in the formation of two highly diagnostic
ions, 0,2A5 for LNFDH-Ia and LNFDH-Ib and 0,1A4/Y3R” for LNFDH-
II, that allow differentiation of LNDFH-II from the Ia and Ib
isomers. Moreover, another series of fragment ions (labeled 3,
C3R ions) permit differentiation of the LNDFH-Ia and -Ib isomers
from LNDFH-II. For example, the C4 ion can be used to
distinguish LNDFH-Ia and -Ib from LNDFH-II, whereas the C3R
and C3R/Y3ꢀ” ions are useful for discrimination of LNDFH-II from
-Ia and -Ib.
7. The CID collision activation voltage (which influences the ion
collision energies and internal energy deposition) or the number
of UV laser pulses (which corresponds to total energy deposition)
was varied while monitoring the total abundances of all reducing
end fragment ions containing the fluorophore and all nonreducing
end fragment ions compared to the abundance of the precursor
ions (based on peak areas). Figure 7A displays the energy-variable
CID fragmentation trends, which show a striking dominance of
reducing end fragments containing the fluorophore as the CID
voltage is raised. A small portion of nonreducing end fragment
ions are observed but remain less than 5% throughout the range
of CID collision energies. In contrast, the UVPD data acquired
as a function of the number of laser pulses for this same AMC-
derivatized oligosaccharide indicate that the nonreducing end
fragment ions are dominant with the fluorophore-containing
reducing end fragments representing less than 5% of the total
abundance (Figure 7B). Interestingly, the pulse-variable UVPD
shows a significant abundance of reducing end fragment ions from
one to three pulses, which indicates that these ions are formed
but decompose upon exposure to subsequent laser pulses. In fact,
the single-pulse UVPD spectrum actually affords a 4:1 ratio of
reducing end to nonreducing end fragment ions, albeit at low total
abundance, which is similar to the distribution observed from the
CID data in which the reducing end fragment ions are consistently
more dominant. In contrast, multiple-pulse UVPD produces more
nonreducing fragment ions, which is attributed to the efficient
secondary dissociation of all fluorophore-containing fragments
(i.e., the reducing end fragments), which are capable of further
photon absorption and dissociation upon subsequent laser pulses
during UV activation. The secondary dissociation of the primary
fluorophore-containing fragment ions in turn creates a combination
of observed internal fragment ions and nonreducing end fragment
A summary of the two main categories of ions produced upon
CID and UVPD of [AMC-LNDFH-II + Na]+ is shown in Figure
5192 Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

Figure 6. ESI-MS/MS spectra of (A) [LNDFH-Ia + Na + AMC]+, (B) [LNDFH-Ib + Na + AMC]+, and (C) [LNDFH-II + Na + AMC]+ by UVPD
with 20 pulses at 60 mJ/pulse for all three spectra. The triangle symbol (3) represents unique fragments useful in isomeric differentiation. Loss
of the fluorophore reagent by cross-ring cleavage of the reducing sugar is labeled -AMC* and -AMC′′, resulting in formation of 0,1A- and
0,2A-type fragment ions, respectively. Loss of the fluorophore reagent and reducing sugar glucose is labeled -AMC+Glc. A star symbol (f) is
used to signify the precursor ion.
ions, as well as fragment ions in the low m/z range that are not
detectable. This highly efficient secondary dissociation of the
fluorophore-containing reducing end fragment ions upon UVPD
ultimately results in the observed MS/MS spectra, which are
dominated by nonreducing end fragments and thus complemen-
tary to the CID mass spectra.
and panels B-E in Figure 8 show the UVPD spectra obtained
from the precursor [LNDFH-Ia + Na + fluorophore]+ for
fluorophores 6-AQ, 2-AB, AMAC, and AMC, respectively.
Different degrees of magnification were applied to each
spectrum to emphasize the similarities in the resulting frag-
mentation pathways irrespective of the fluorophore. As noted
in earlier sections, the fragment ions produced upon UVPD
mainly stem from neutral losses that incorporate the fluoro-
phore tag. Based on the specific fluorophore tag, the relative
UVPD efficiencies of the LNDFH-Ia ions are summarized in
decreasing order as follows: 6-AQ [Na+] (65.0%); AMAC
(48.6%); AMC (41.9%); 6-AQ [H+] (21.0%); and 2-AB (10.5%). A
similar trend is observed for other fluorophore-labeled oli-
gosaccharides, and these data support 6-AQ as the best reagent
for UVPD strategies.
Figure 8A shows the UVPD spectrum of protonated 6-AQ-
LNDFH-Ia for comparison to that of sodium-cationized 6-AQ-
LNDFH-Ia (Figure 8B). The UVPD spectra of protonated oli-
gosaccharides actually afford rather convoluted branching structure
information due to fructose rearrangements, which have previ-
ously been observed upon collision-induced dissociation of pro-
tonated reductively aminated oligosaccharides.62 Franz et al.
proposed a two-step proton-catalyzed mechanism that facilitates
a long-range glycosyl-transfer reaction for protonated oligosac-
charides that are fluorescently labeled.62 This feature makes
structural elucidation of protonated oligosaccharides difficult, but
fortunately, the sodium-cationized species follow predictable
fragmentation routes and thus are preferred for MS/MS applica-
tions. Moreover, the difference in UVPD efficiencies for the
Influence of Fluorophore on UVPD Efficiency. The ideal
fluorophores for oligosaccharide analysis will allow both
fluorescence quantification and UVPD characterization, in
addition to increasing the hydrophobicity of the targeted
oligosaccharides for improved separation by HPLC.61 For MS/
MS identification by UVPD, the fluorophore should not only
undergo efficient photon absorption but also promote diagnostic
dissociation of the oligosaccharides, as opposed to less mean-
ingful pathways such as cleavage of the fluorophore tag.
Therefore, the relative PD efficiency afforded by incorporation
of different fluorophore labels is a key performance parameter.
Absorption and fluorescence properties of fluorophore-labeled
oligosaccharides in solution may differ dramatically from the
analogous data for gas-phase ions due to the impact of solvation
and charge state, and thus, UVPD dissociation efficiencies are
best predicted from energy-variable UVPD measurements in
the gas phase. For these experiments, the relative extent of
UVPD is measured for an oligosaccharide as a function of the
fluorophore tag for a fixed set of UVPD parameters (e.g., 20
laser pulses at 60 mJ/pulse). Examples of the resulting
comparative UVPD spectra are shown in Figure 8 for LNDFH-
Ia derivatized with four different fluorophores. Figure 8A shows
the UPVD spectrum obtained for protonated 6-AQ-LNDFH-Ia,
(61) Delaney, J.; Vouros, P. Rapid Commun. Mass Spectrom. 2001, 15, 325–
(62) Franz Andreas, H.; Lebrilla Carlito, B. J. Am. Soc. Mass Spectrom. 2002,
334
.
13, 325–337.
Analytical Chemistry, Vol. 80, No. 13, July 1, 2008 5193

instead of B-type ions upon UV irradiation is suggestive of greater
internal energy deposition than that which occurs during colli-
sional activation and is supported by the fact that C-type fragment
ion formation is more commonly observed in high-energy CID
experiments.22 Based on this observation, it is probable that the
labeled (Man)3-5 fragment ions in the UVPD spectra could be
the result of glycosidic bond cleavages that lead to C-/Z-ions
instead of B-/Y-ions, which result in isomeric fragment overlap.
For example, in Figure 9B, “B3/Y3ꢀ or 4R” could in fact be created
from “C3/Z3ꢀ or 4R”, in both cases resulting in ions containing four
mannose units (Man)4. Due to this uncertainty in identification,
many of these fragments are simply labeled (Man)x where x is
the number of remaining mannose units. This data demonstrates
the potential for characterization of glycans released from glyco-
proteins by implementing UVPD to probe key diagnostic fragment
ions.
Fluorophore-Assisted Electron Photodetachment Dis-
sociation. The attachment of fluorophores to oligosaccharides
also offers the option of pursuing electron photodetachment
dissociation42 strategies instead of UVPD. In EPD, negatively
charged ions undergo electron detachment upon laser irradia-
tion to produce charge-reduced radical ions. The charged-
reduced radical ions may exhibit different dissociation routes
upon subsequent collisional activation than conventional closed-
shell anions. For oligosaccharides, EDD, a method in which
deprotonated molecules stored in an FTICR mass spectrometer
are irradiated with electrons, thus causing electron detachment
and ion dissociation, results in additional cross-ring cleavages
such as3,5A-,1,5A-,1,5X-, and3,5X-type ions, which can greatly
assist in linkage characterization.29 In contrast to EDD, EPD
has not been previously reported for the analysis of oligosac-
charides, in large part because underivatized oligosaccharides
do not efficiently absorb UV photons. For the present work,
an acidic fluorophore was used to derivatize the oligosaccha-
rides in order to ensure the efficient formation of doubly
charged negative ions upon ESI. Examples of the resulting EP
and EPD spectra are shown in Figure 10 for LNDFH-II
derivatized with AGA. Figure 10A shows the electron photo-
detachment spectrum obtained upon UV irradiation of the
doubly deprotonated precursor, resulting in conversion of [(M
+ AGA) - 2H]2- ions to charge-reduced [(M + AGA) - 2H]-
radical species. Confirmation that the charge-reduced products
formed upon the UV irradiation are in fact radical species is
obtained by expansion of the molecular ion region and
comparison to the molecular ion region of conventional depro-
tonated LNDFH-II (Figure 10D). The radical species appears
1 Da lower in mass than the conventional deprotonated species,
which is consistent with its origins from a doubly deprotonated
precursor. When the [(M + AGA) - 2H]- radical ion is
subsequently subjected to CID, the EPD spectrum in Figure
10B is obtained. The fragmentation pattern of the radical ion
is dramatically different from that of the conventional singly
deprotonated precursor (Figure 10C). The CID spectrum of
the conventional deprotonated oligosaccharide in Figure 10C
displays mainly Y-type fragment ions, whereas the EPD
spectrum in Figure 10B reveals Y-, Z-, and cross-ring cleavage
X-type fragment ions. These striking differences in the frag-
mentation patterns of the radical ions and deprotonated species
Figure 7. Relative peak area abundance of the precursor, sum of
nonreducing end fragments, and sum of reducing end fragments
versus (A) CID collision activation voltage and (B) the number of laser
pulses (355 nm, 60 mJ/pulse at 10 Hz) of [LNDFH-II + Na + AMC]+.
protonated (21%) versus sodium-cationized (65%) LNDHFH-Ia
species is significant, which also argues against using the
protonated species for UVPD applications.
MS/MS of High-Mannose Glycans Released from Ribo-
nuclease B. Glycans can also be derivatized to undergo efficient
UVPD after their enzymatic release from glycoproteins and
subsequent reductive amination. Ribonuclease B is a good model
protein due to its relatively simple glycan composition that
incorporates five variants of the high-mannose N-linked glycan
(GlcNAc2Man5-9). These glycans were released from the glyco-
protein by enzymatic treatment with PNGase F and then deriva-
tized with 6-AQ. The two most abundant of these glycans,
GlcNAc2Man5 and GlcNAc2Man6, were subjected to CID and
UVPD with the resulting MS/MS spectra shown in Figure 9.
Interestingly, unlike the LNFP and LNDFH oligosaccharides,
these glycans have a tendency to lose the fluorophore upon
activation, yielding B-type fragment ions by CID (Figure 9A and
C). Consequently, these CID spectra afford information more
similar to their UVPD counterparts (Figure 9B and D) than noted
for the earlier CID versus UVPD comparisons for the oligosac-
charides; however, there are still several distinct differences. In
contrast to the CID mass spectra, the UVPD spectra display not
only 0,1A- and 0,2A-type fragment ions but also dominant C-type
ions that were not observed in the corresponding CID spectra,
as opposed to the more prevalent B-type fragment ions seen in
the CID mass spectra. The prevalent formation of C-type ions
5194 Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

Figure 8. ESI-MS/MS of [LNDFH-Ia + Na + label]+ comparing the relative dissociation efficiencies of different fluorescent labels in parentheses
including (A) 6-AQ [H+] (21.0%), (B) 6-AQ [Na+] (65.0%), (C) 2-AB (10.5%), (D) AMAC (48.6%), and (E) AMC (41.9%) by UVPD using 20
pulses for all five spectra. A star symbol (f) is used to signify the precursor ion. Loss of the fluorophore reagent by cross-ring cleavage of the reducing
sugar is labeled -FL* and -FL′′ (where FL is a generic term used to represent 6-AQ, AMAC, AMC, or 2-AB), resulting in formation of 0,1A- and
0,2A-type fragment ions, respectively. Loss of the fluorophore reagent and reducing sugar glucose is labeled -FL+Glc. Various degrees of magnification
are shown for each spectrum over the indicated mass range to scale up the fragment ions to allow easier visual comparison.
Figure 9. ESI-MS/MS spectra of [GlcNAc2Man5 + Na + 6-AQ]+ by (A) CID and (B) UVPD with 20 pulses and [GlcNAc2Man6 + Na + 6-AQ]+
by (C) CID and (D) UVPD with 20 pulses. A star symbol (f) is used to signify the precursor ion. Loss of the fluorophore reagent by cross-ring
cleavage of the reducing sugar is labeled -6-AQ* and -6-AQ′′, resulting in formation of 0,1A- and 0,2A-type fragment ions, respectively. Loss
of the fluorophore reagent and reducing sugar is labeled -6-AQ+GlcNAc. The magnification scale bars apply only to (B) and (D) over the
indicated mass range.
are under further investigation and may lead to development
of EPD as a promising tandem mass spectrometric method for
characterization of oligosaccharides. Derivatization of the
oligosaccharides with the nonacidic fluorophores shown in
Analytical Chemistry, Vol. 80, No. 13, July 1, 2008 5195

Figure 10. Electron photodetachment dissociation of LNDFH-II derivatized with AGA, where (A) shows the EPD spectrum upon irradiation of
doubly deprotonated LNDFH-II using 20 laser pulses resulting in production of the radical species [M + AGA - 2H] -. (B) CID of the resulting
radical ion; (C) CID of [LNDFH-II + AGA -H]-. (D) Expanded regions around the precursor ions show the 1-Da mass difference between the
radical ion and conventional closed-shell deprotonated species. A star symbol (f) is used to signify the precursor ion.
Figure 1 did not lead to efficient production of negative ions in
the negative ESI mode, nor to successful EPD.
ages not typically observed for conventional closed-shell depro-
tonated species nor sodium-cationized complexes.
CONCLUSION
ACKNOWLEDGMENT
Fluorescently labeled oligosaccharides undergo efficient UVPD
at 355 nm, which leads to production of a complementary series
of fragment ions compared to CID. Multiple-pulse UVPD results
in extensive secondary dissociation and primarily formation of
fragment ions from the nonreducing end in contrast to CID, which
typically results in reducing-end fragment ions that retain the
appended fluorophore. UVPD shows promise for facile isomeric
differentiation of oligosaccharides, especially in combination with
CID experiments for comprehensive structural characterization.
This method has been extended to a model glycoprotein, ribo-
nuclease B, based on enzymatic release of the glycans, subsequent
derivatization, and UVPD analysis. The first EPD results for
fluorophore-derivatized oligosaccharides showcase cross-ring cleav-
Funding from the Welch Foundation (F1155), the National
Science Foundation (CHE-0718320) and an NSF/IGERT fellowship
to J.J.W. (D6E-03333080) are gratefully acknowledged.
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in text includes two supple-
mental figures: CID MS/MS spectra of sodium-cationized LNFP-
I, -II, -III, and -V, as well as LNDFH-Ia, -Ib, and -II. This material
is available free of charge via the Internet at http://pubs.acs.org.
Received for review February 14, 2008. Accepted April 10,
2008.
AC800315K
5196 Analytical Chemistry, Vol. 80, No. 13, July 1, 2008