Infrared multiphoton dissociation of duplex DNA/drug complexes in a quadrupole ion trap

Noncovalent duplex DNA/drug complexes formed between one of three 14-base pair non-self-complementary duplexes with variable GC content and one of eight different DNA-interactive drugs are characterized by infrared multiphoton dissociation (IRMPD), and the resulting spectra are compared to conventional collisionally activated dissociation (CAD) mass spectra in a quadrupole ion trap mass spectrometer. IRMPD yielded comparable information to previously reported CAD results in which strand separation pathways dominate for complexes containing the more AT-rich sequences and/or minor groove binding drugs, whereas drug ejection pathways are prominent for complexes containing intercalating drugs and/or duplexes with higher GC base content. The large photoabsorptive cross section of the phosphate backbone at 10.6 mum promotes highly efficient dissociation within short irradiation times (<2 ms at 50 W) or using lower laser powers and longer irradiation times (<15 W at 15 ms), activation times on par with or shorter than standard CAD experiments. This large photoabsorptivity leads to a controllable ion activation method which can be used to produce qualitatively similar spectra to CAD while minimizing uninformative base loss dissociation pathways or instead be tuned to yield a high degree of secondary fragmentation. Additionally, the low-mass cutoff associated with conventional CAD plays no role in IRMPD, resulting in richer MS/MS information in the low m/z region. IRMPD is also used for multiadduct dissociation in order to increase MS/MS sensitivity, and a two-stage IRMPD/IRMPD method is demonstrated as a means to give specific DNA sequence information that would be useful when screening drug binding by mixtures of duplexes.     

Infrared multiphoton dissociation and electron-induced dissociation as alternative MS/MS strategies for metabolite identification

A major challenge encountered in mass spectrometric metabolite analysis is the identification and structural characterization of metabolites. Fourier transform ion cyclotron resonance mass spectrometry is a valuable technique for metabolite structural determination because it provides accurate masses and allows for multiple MS/MS fragmentation strategies, including infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID). Collision activated dissociation (CAD) is currently the most commonly used MS/MS technique for metabolite structural characterization. In contrast, IRMPD and EID have had very limited, if any, application for metabolite characterization. Here, we explore IRMPD and EID of phosphate-containing metabolites and compare the resulting fragmentation patterns to those of CAD. Our results show that CAD, IRMPD, and EID provide complementary structural information for phosphate-containing metabolites. Overall, CAD provided the most extensive fragmentation for smaller (<600 Da) phosphate-containing metabolites; however, IRMPD generated more extensive fragmentation for larger (>600 Da) phosphate-containing metabolites, particularly for species containing increased numbers of phosphate groups. EID generally provided complementary fragmentation to CAD and showed extensive fragmentation with relatively evenly abundant product ions, regardless of metabolite size. However, EID fragmentation efficiency is lower than those of CAD and IRMPD.     

Infrared multiphoton dissociation for quantitative shotgun proteomics

We modified a dual-cell linear ion trap mass spectrometer to perform infrared multiphoton dissociation (IRMPD) in the low-pressure trap of a dual-cell quadrupole linear ion trap (dual-cell QLT) and perform large-scale IRMPD analyses of complex peptide mixtures. Upon optimization of activation parameters (precursor q-value, irradiation time, and photon flux), IRMPD subtly, but significantly, outperforms resonant-excitation collisional-activated dissociation (CAD) for peptides identified at a 1% false-discovery rate (FDR) from a yeast tryptic digest (95% confidence, p = 0.019). We further demonstrate that IRMPD is compatible with the analysis of isobaric-tagged peptides. Using fixed QLT rf amplitude allows for the consistent retention of reporter ions, but necessitates the use of variable IRMPD irradiation times, dependent upon precursor mass to charge (m/z). We show that IRMPD activation parameters can be tuned to allow for effective peptide identification and quantitation simultaneously. We thus conclude that IRMPD performed in a dual-cell ion trap is an effective option for the large-scale analysis of both unmodified and isobaric-tagged peptides.     

Infrared multiphoton dissociation for enhanced de novo sequence interpretation of N-terminal sulfonated peptides in a quadrupole ion trap

Infrared multiphoton dissociation (IRMPD) of N-terminal sulfonated peptides improves de novo sequencing capabilities in a quadrupole ion trap mass spectrometer. Not only does IRMPD promote highly efficient dissociation of the N-terminal sulfonated peptides but also the entire series of y ions down to the y(1) fragment may be detected due to alleviation of the low-mass cutoff problem associated with conventional collisional activated dissociation (CAD) methods in a quadrupole ion trap. Commercial de novo sequencing software was applied for the interpretation of CAD and IRMPD MS/MS spectra collected for seven unmodified peptides and the corresponding N-terminal sulfonated species. In most cases, the additional information obtained by N-terminal sulfonation in combination with IRMPD provided significant improvements in sequence identification. The software sequence tag results were combined with a commercial database searching algorithm to interpret sequence information of a tryptic digest on alpha-casein s1. Energy-variable CAD studies confirmed a 30-40% reduction in the critical energies of the N-terminal sulfonated peptides relative to unmodified peptides. This reduction in dissociation energy facilitates IRMPD in a quadrupole ion trap.     

Silver complexation and tandem mass spectrometry for differentiation of isomeric flavonoid diglycosides

For detection and differentiation of isomeric flavonoids, electrospray ionization mass spectrometry is used to generate silver complexes of the type (Ag + flavonoid)+. Collisionally activated dissociation (CAD) of the resulting 1:1 silver/flavonoid complexes allows isomer differentiation of flavonoids. Eighteen flavonoid diglycosides constituting seven isomeric series are distinguishable from each other based on the CAD patterns of their silver complexes. Characteristic dissociation pathways allow identification of the site of glycosylation, the type of disaccharide (rutinose versus neohesperidose), and the type of aglycon (flavonol versus flavone versus flavanone). This silver complexation method is more universal than previous metal complexation methods, as intense silver complexes are observed even for flavonoids that lack the typical metal chelation sites. To demonstrate the feasibility of using silver complexation and tandem mass spectrometry to characterize flavonoids in complex mixtures, flavonoids extracted from grapefruit juice are separated by high-performance liquid chromatography and analyzed via a postcolumn complexation ESI-MS/MS strategy. Diagnostic fragmentation pathways of the silver complexes of the individual eluting flavonoids allow successful identification of the six flavonoids in the extract.     

Infrared multiphoton dissociation in an external ion reservoir.

In this work we present a novel scheme for performing infrared multiphoton dissociation (IRMPD) external to the mass analyzer in an external ion reservoir consisting of an rf-only multipole and a pair of electrostatic lens elements. Ions generated by electrospray ionization (ESI) are accumulated in an rf-only hexapole and dissociated by irradiation at 10.6 microns from a CW CO2 laser in the source region of the mass spectrometer. This scheme is unique from other IRMPD schemes as dissociation occurs in a spatially distinct region of the spectrometer and is independent of the mass spectrometry platform used to analyze the fragment ions. The effectiveness of the technique is demonstrated with ESI IRMPD FTICR mass spectrometry of a 20-mer phosphorothioate oligonucleotide. A comparison of the external IRMPD scheme with nozzle-skimmer dissociation and conventional in-cell IRMPD reveals a significant improvement in signal-to-noise ratio and fragment yield, particularly for larger, more highly charged fragment ions.     

Consecutive ion activation for top down mass spectrometry: improved protein sequencing by nozzle-skimmer dissociation

Mass spectra produced by nozzle-skimmer dissociation (NSD) have been little used in the past for structural characterization. NSD cannot be used on mass-separated ions (MS/MS), and for electrosprayed protein ions, previous NSD spectra showed backbone cleavages similar to those from energetic methods such as collisionally activated dissociation (CAD) or infrared multiphoton dissociation (IRMPD). However, our experimental configuration with Fourier transform (FT) MS makes possible three consecutive steps of NSD ion activation: thermal in the entrance capillary and collisional in both the nozzle-skimmer (N-S) region and the region after the skimmer before the quadrupole entrance lens (S-Q). In the high-pressure N-S region of adjustable path length, ions undergo high-frequency, low-energy collisions to rupture weak noncovalent or covalent bonds, with these "denatured" products then subjected to high-energy collisions in the low-pressure S-Q region to cleave strong backbone bonds. These NSD spectra, plus those from variable capillary thermal activation, of 8+ to 11+ ubiquitin ions electrosprayed from denatured solution show backbone cleavages between 74 of 75 amino acid pairs, vs 66 for CAD and 50 for IRMPD in the FTMS cell. Thermal activation by the inlet capillary of the newly desolvated 6+, 7+ ubiquitin ions from electrospraying the native conformer increases the NSD yield from 8% at 56 degrees C to 96% at 76 degrees C, but with little change in product branching ratios; this capillary heating has no effect on CAD or IRMPD of these ions collected in the FTMS cell. Ion desolvation with its concomitant H-bond strengthening appears to produce a transiently stable conformer whose formation can be prevented by capillary heating. The far more complex and stable noncovalent tertiary structures of large protein ions in the gas phase have made MS/MS difficult; initial inhibition of tertiary structure formation with immediate NSD ("prefolding dissociation") appears promising for the top down characterization of a 200-kDa protein.     

Probing ligand binding to duplex DNA using KMnO4 reactions and electrospray ionization tandem mass spectrometry

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) strategy employing the thymine-selective KMnO4 oxidation reaction to detect conformational changes and ligand binding sites in noncovalent DNA/drug complexes is reported. ESI-MS/MS is used to detect specific mass shifts of the DNA ions that are associated with the oxidation of thymines. This KMnO4 oxidation/ESI-MS/MS approach is an alternative to conventional gel-based oxidation methods and affords excellent sensitivity while eliminating the reliance on radiolabeled DNA. Comparison of single-strand versus duplex DNA indicates that the duplexes exhibit a significant resistance to the reaction, thus confirming that the oxidation process is favored for unwound or single-strand regions of DNA. DNA complexes containing different drugs including echinomycin, actinomycin-D, ethidium bromide, Hoechst 33342, and cis-C1 were subjected to the oxidation reaction. Echinomycin, a ligand with a bisintercalative binding mode, was found to induce the greatest KMnO4 reactivity, while Hoechst 33342, a minor groove binder, caused no increase in the oxidation of DNA. The oxidation of echinomycin/DNA complexes containing duplexes with different sequences and lengths was also assessed. Duplexes with thymines closer to the terminal ends of the duplex demonstrated a greater increase in the degree of oxidation than those with thymines in the middle of the sequence. Collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) experiments were used to determine the site of oxidation based on oligonucleotide fragmentation patterns.     

Differentiation of closely related isomers: application of data mining techniques in conjunction with variable wavelength infrared multiple photon dissociation mass spectrometry for identification of glucose-containing disaccharide ions

Data mining algorithms have been used to analyze the infrared multiple photon dissociation (IRMPD) patterns of gas-phase lithiated disaccharide isomers irradiated with either a line-tunable CO(2) laser or a free electron laser (FEL). The IR fragmentation patterns over the wavelength range of 9.2-10.6 μm have been shown in earlier work to correlate uniquely with the asymmetry at the anomeric carbon in each disaccharide. Application of data mining approaches for data analysis allowed unambiguous determination of the anomeric carbon configurations for each disaccharide isomer pair using fragmentation data at a single wavelength. In addition, the linkage positions were easily assigned. This combination of wavelength-selective IRMPD and data mining offers a powerful and convenient tool for differentiation of structurally closely related isomers, including those of gas-phase carbohydrate complexes.     

Infrared multiple photon dissociation spectroscopy and computational studies of O-glycosylated peptides

The infrared multiple photon dissociation (IRMPD) spectra of O-glycosylated peptides in the gas phase were studied in the IR scanning range of 5.7-9.5 μm. Fragmentation of protonated and sodiated O-glycopeptides was investigated using electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with a free electron laser (FEL). FEL is used in the IRMPD technique as a tunable IR light source. In the IRMPD spectroscopic analysis of the protonated O-glycopeptide, fragment ions of the b/y and B/Y types were observed in the range of 5.7-9.5 μm, corresponding to the cleavage of the backbone in the parent amino acid sequence and glycosyl bonds, whereas the spectra of the sodiated glycopeptide showed major peaks of photoproducts of the B/Y type in the range of 8.4-9.5 μm. The IRMPD spectra of the O-glycopeptides were compared with simulated IR spectra for the structures obtained from the molecular dynamics.     

Determination of phospholipid regiochemistry by Ag(I) adduction and tandem mass spectrometry

Collision-activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) of Ag-adducted phospholipids were investigated as structural tools. Previously, determination of the acyl chains at the two phospholipid esterification sites has been performed based on the R(1)COO(-)/R(2)COO(-) ratio in negative ion mode CAD tandem mass spectrometry. However, the observed product ion ratio is dependent on the extent of unsaturation of the fatty acyl group at sn-2 as well as on the total chain length. Similarly, in positive ion mode CAD with/without alkaline or alkaline earth metal adduction, the ratio of product ions resulting from either R(1)COOH or R(2)COOH neutral losses is dependent on the nature of the phospholipid polar headgroup. Ag(+) ion chromatography, in which silver ions are part of the stationary phase, can provide information on double bond number/distribution as well as double bond configuration (cis/trans) because of interaction between Ag(+) ions and olefinic π electrons of fatty acids and lipids. We hypothesized that interactions between double bonds and Ag(+) may be utilized to also reveal phospholipid esterification site information in tandem mass spectrometry. CAD and IRMPD of Ag-adducted phospholipids with unsaturated fatty acids (R(x)COOH, x = 1 or 2) provided characteristic product ions, [R(x)COOH + Ag](+), and their neutral losses. The characteristic product ions and their abundances do not depend on the type of polar headgroup or the number of double bonds of unsaturated acyl chains. Tandem mass spectrometry of Cu-adducted phospholipids was also performed for comparison based on the Lewis acid and base properties of Cu(+) and phospholipid double bonds, respectively.     

Electron capture dissociation of oligosaccharides ionized with alkali, alkaline Earth, and transition metals

We extend the application of electron capture dissociation (ECD) (which requires at least two charges) to oligosaccharides without basic functionalities by utilizing alkali, alkaline earth, and transition metals (Na+, K+, Ca2+, Ba2+, Mg2+, Mn2+, Co2+, and Zn2+) as charge carriers in electrospray ionization. Both linear and branched oligosaccharides were examined, including maltoheptoase, p-lacto-N-hexaose, and an N-linked glycan from human alpha1-acid glycoprotein. For comparison, infrared multiphoton dissociation (IRMPD) was also applied to all oligosaccharide species. We show that, for certain metal-adducted oligosaccharides, particularly maltoheptaose, cross-ring cleavage, which can provide saccharide linkage information, is the dominant fragmentation pathway in ECD. By contrast, glycosidic cleavages dominate in IRMPD although cross-ring fragmentation was also observed to varying degrees depending on metal ion type. The branched N-linked glycan did not fragment as easily following ECD compared to the linear oligosaccharides, presumably due to intramolecular noncovalent interactions. However, this limitation was partially overcome with a combined ECD/IRMPD approach (activated ion ECD). For all metal-adducted oligosaccharides, complementary structural information was obtained with ECD as compared to IRMPD. Our results demonstrate that ECD of metal-adducted oligosaccharides is a valuable tool for structural characterization of oligosaccharides.     

Hybrid activation methods for elucidating nucleic acid modifications

Hybrid tandem mass spectrometry (MS/MS) techniques combining electron transfer (ET) and collision activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), or ultraviolet photodissociation (UVPD) were implemented and evaluated for the characterization of a series of oligonucleotides and oligoribonucleotides, including both native single strands and single strands containing platinated, phosphorothioated, and 2'-O-methylated modification sites. ET-IRMPD and ET-UVPD of oligodeoxynucleotides and oligoribonucleotides resulted in rich fragmentation with respect to production of w, a, z, and d ions for DNA, and c, y, w, a-B, d, and z ions for RNA, with many product ions retaining the modification and thus allowing site specific identification. ET-IRMPD caused more extensive secondary dissociation of the ions, in addition to a broader distribution of detectable sequence ions attributed to using a lower mass cutoff. ET-UVPD promoted higher energy fragmentation pathways and created the most diverse MS/MS spectra. The numerous products generated by the hybrid MS/MS techniques resulted in specific and extensive backbone cleavages which allowed the modification sites of multiply modified oligonucleotides to be elucidated.     

Interrogation of N-Linked oligosaccharides using infrared multiphoton dissociation in FT-ICR mass spectrometry

The structural elucidation of oligosaccharides remains a major challenge. Mass spectrometry provides a rapid and convenient method for structural elucidation based on tandem mass spectrometry. Ions commonly are selected and subjected to collision-induced dissociation (CID) to obtain structural information. Unfortunately, N-linked oligosaccharides are relatively large compounds and are not readily fragmented using CID. In this report, we illustrate the use of infrared multiphoton dissociation (IRMPD) to obtain structural information for large N-linked oligosaccharides. The IRMPD and CID behavior of oligosaccharides were compared for high-mannose-type oligosaccharides. Fragmentation that could not be obtained through conventional CID in Fourier transform ion cyclotron resonance mass spectrometry was observed with N-linked oligosaccharides. O-Linked and N-linked glycans of similarly large sizes were compared. It was found that internal cross-ring cleavages were observed only for N-linked oligosaccharides. The mannose branch points of N-linked oligosaccharides are apparently more susceptible to cross-ring cleavages.     

Thermally assisted infrared multiphoton photodissociation in a quadrupole ion trap

Thermally assisted infrared multiphoton photodissociation (TA-IRMPD) provides an effective means to dissociate ions in the quadrupole ion trap mass spectrometer (QITMS) without detrimentally affecting the performance of the instrument. IRMPD can offer advantages over collision-induced dissociation (CID). However, collisions with the QITMS bath gas at the standard pressure and ambient temperature cause IR-irradiated ions to lose energy faster than photons can be absorbed to induce dissociation. The low pressure required for IRMPD (< or = 10(-5) Torr) is not that required for optimal performance of the QITMS (10(-3) Torr), and sensitivity and resolution suffer. TA-IRMPD is performed with the bath gas at an elevated temperature. The higher temperature of the bath gas results in less energy lost in collisions of the IR-excited ions with the bath gas. Thermal assistance allows IRMPD to be used at or near optimal pressures, which results in an approximately 1 order of magnitude increase in signal intensity. Unlike CID, IRMPD allows small product ions, those less than about one-third the m/z of the parent ion, to be observed. IRMPD should also be more easily paired with fluctuating ion sources, as the corresponding fluctuations in resonant frequencies do not affect IRMPD. Finally, while IR irradiation nonselectively causes dissociation of all ions, TA-IRMPD can be made selective by using axial expansion to move ions away from the path of the laser beam.     

Infrared multiphoton dissociation with a hollow fiber waveguide

A novel scheme for performing infrared multiphoton dissociation (IRMPD) is presented in which a hollow fiber waveguide (HFWG) is used to transmit IR radiation into the ion storage region of a mass spectrometer. Efficient dissociation of oligonucleotide and protein ions is demonstrated on an ESI-FTICR instrument in which IRMPD is performed in the external ion reservoir and on a quadrupole ion trap. Using a simple optical scheme consisting of a single focusing lens and an x, y translator, the 10.6-microm IR laser beam, initially 3.5 mm in diameter, is focused into the vacuum-sealed HFWG. The small internal diameter and the high transfer efficiency of the waveguide allow IR radiation of high power density to be employed for IRMPD. In studies performed on a quadrupole ion trap, a 500-microm-i.d. waveguide was used as a medium to transmit IR radiation directly through a 700-microm orifice in the ring electrode. Efficient IRMPD of both a 12-mer oligonucleotide and the protein melittin were performed at laser powers of 0.5 and 3.2 W, respectively.     

Electron capture dissociation and infrared multiphoton dissociation MS/MS of an N-glycosylated tryptic peptic to yield complementary sequence information

Glycoproteins are a functionally important class of biomolecules for which structural elucidation presents a challenge. Fragmentation of N-glycosylated peptides, employing collisionally activated dissociation, typically yields product ions that result from dissociation at glycosidic bonds, with little occurrence of dissociation at peptide backbone sites. We have applied two dissociation techniques, electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD), in a 7-T Fourier transform ion cyclotron resonance mass spectrometer, in the investigation of an N-glycosylated peptide from an unfractionated tryptic digest of the lectin of the coral tree, Erythrina corallodendron. ECD provided c and z. ions derived from the peptide backbone, with no observed loss of sugars. Cleavage at 11 of 15 backbone amine bonds was observed. The lack of cleavage at sites located close to the glycosylated asparagine residue may result from steric blocking by the glycan. IRMPD provided abundant fragment ions, primarily through dissociation at glycosidic linkages. The monosaccharide composition and the presence of three glycan branch sites could be determined from the IRMPD fragments. The two types of spectra, obtained with the same instrument, thus provide complementary structural information about the glycopeptide. The current result extends the applicability of ECD for glycopeptide analysis to N-glycosylated peptides and to peptides containing branched, highly substituted glycans.     

Infrared multiphoton dissociation in quadrupole time-of-flight mass spectrometry: top-down characterization of proteins

The first implementation of infrared multiphoton dissociation (IRMPD) for a hybrid quadrupole time-of-flight (QqTOF) mass spectrometer is reported. Ions were trapped in the radio frequency-only quadrupole (q2), which normally serves as a collision cell, and irradiated by a continuous CO2 IR laser. The laser beam was introduced coaxially with the quadrupoles in order to maximize overlap with the ion path. The resolution of the TOF mass analyzer allowed direct charge state determination for fragments smaller than 7 kDa. For larger fragments, the charge state could be assigned using the multiple losses of water, characteristic for IRMPD of proteins. The analytical performance is demonstrated by top-down sequencing of several representative proteins (equine myoglobin, bovine casein, and human insulin and chaperonin 10). Various post-translational modifications such as phosphorylation, acetylation, formation of disulfide bridges, and removal of N-terminal methionine followed by acetylation are detected and characterized. The utility of IRMPD for the analysis of biological samples is demonstrated in a study of a recently identified potential marker for endometrial cancer, chaperonin 10.     

Combined electron capture and infrared multiphoton dissociation for multistage MS/MS in a Fourier transform ion cyclotron resonance mass spectrometer

We have mounted a permanent on-axis dispenser cathode electron source inside the magnet bore of a 9.4-T Fourier transform ion cyclotron resonance mass spectrometer. This configuration allows electron capture dissociation (ECD) to be performed reliably on a millisecond time scale. We have also implemented an off-axis laser geometry that enables simultaneous access to ECD and infrared multiphoton dissociation (IRMPD). Optimum performance of both fragmentation techniques is maintained. The analytical utility of performing either ECD or IRMPD on a given precursor ion population is demonstrated by structural characterization of several posttranslationally modified peptides: IRMPD of phosphorylated peptides results in few backbone (b- and y-type) cleavages, and product ion spectra are dominated by neutral loss of H3PO4. In contrast, ECD provides significantly more backbone (c- and z*-type) cleavages without loss of H3PO4. For N-glycosylated tryptic peptides, IRMPD causes extensive cleavage of the glycosidic bonds, providing structural information about the glycans. ECD cleaves all backbone bonds (except the N-terminal side of proline) in a 3-kDa glycopeptide with no saccharide loss. However, only a charge-reduced radical species and some side chain losses are observed following ECD of a 5-kDa glycopeptide from the same protein. An MS3 experiment involving IR laser irradiation of the charge-reduced species formed by electron capture results in extensive dissociation into c- and z-type fragment ions. Mass-selective external ion accumulation is essential for the structural characterization of these low-abundance (modified) peptides.     

Infrared multiphoton dissociation of O-linked mucin-type oligosaccharides

Oligosaccharides are known to play important roles in many biological processes. In the study of oligosaccharides, collision-induced dissociation (CID) is the most common dissociation method to elucidate the sequence and connectivity. However, a disadvantage of CID is the decrease in both the degree and efficiency of dissociation with increasing mass. In the present study, we have successfully performed infrared multiphoton dissociation (IRMPD) on 39 O-linked mucin-type oligosaccharide alditols (both neutral and anionic). CID and IRMPD spectra of several oligosaccharides were also compared. They yielded nearly identical fragment ions corresponding to the lowest energy fragmentation pathways. The characteristic fragmentations of structural motifs, which can provide the linkage information, were similarly presented in both CID and IRMPD spectra. Multistage of CID (MS(3) or MS(4)) is commonly needed to completely sequence the oligosaccharides, while IRMPD of the same compounds yielded the fragment ions corresponding to the loss of the first residue to the last residue during a single-stage tandem MS (MS(2)). Finally, it is shown that the fragmentation efficiency of IRMPD increases with the increasing size of oligosaccharides.