Enhanced characterization of singly protonated phosphopeptide ions by femtosecond laser-induced ionization/dissociation tandem mass spectrometry (fs-LID-MS/MS)

To develop an improved understanding of the regulatory role that post-translational modifications (PTMs) involving phosphorylation play in the maintenance of normal cellular function, tandem mass spectrometry (MS/MS) strategies coupled with ion activation techniques such as collision-induced dissociation (CID) and electron-transfer dissociation (ETD) are typically employed to identify the presence and site-specific locations of the phosphate moieties within a given phosphoprotein of interest. However, the ability of these techniques to obtain sufficient structural information for unambiguous phosphopeptide identification and characterization is highly dependent on the ion activation method employed and the properties of the precursor ion that is subjected to dissociation. Herein, we describe the application of a recently developed alternative ion activation technique for phosphopeptide analysis, termed femtosecond laser-induced ionization/dissociation (fs-LID). In contrast to CID and ETD, fs-LID is shown to be particularly suited to the analysis of singly protonated phosphopeptide ions, yielding a wide range of product ions including a, b, c, x, y, and z sequence ions, as well as ions that are potentially diagnostic of the positions of phosphorylation (e.g., ‘a _n+1–98’). Importantly, the lack of phosphate moiety losses or phosphate group ‘scrambling’ provides unambiguous information for sequence identification and phosphorylation site characterization. Therefore, fs-LID-MS/MS can serve as a complementary technique to established methodologies for phosphoproteomic analysis.     

Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic C-Terminal Residue

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H₃PO₄), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H₃PO₄ elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H₃PO₄ upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available.

Comparison of CID,ETD and metastable atom‐activated dissociation (MAD) of doubly and triply charged phosphorylated tau peptides

The fragmentation behavior of the 2+ and 3+ charge states of eleven different phosphorylated tau peptides was studied using collision‐induced dissociation (CID), electron transfer dissociation (ETD) and metastable atom‐activated dissociation (MAD). The synthetic peptides studied contain up to two known phosphorylation sites on serine or threonine residues, at least two basic residues, and between four and eight potential sites of phosphorylation. CID produced mainly b‐/y‐type ions with abundant neutral losses of the phosphorylation modification. ETD produced c‐/z‐type ions in highest abundance but also showed numerous y‐type ions at a frequency about 50% that of the z‐type ions. The major peaks observed in the ETD spectra correspond to the charge‐reduced product ions and small neutral losses from the charge‐reduced peaks. ETD of the 2+ charge state of each peptide generally produced fewer backbone cleavages than the 3+ charge state, consistent with previous reports. Regardless of charge state, MAD achieved more extensive backbone cleavage than CID or ETD, while retaining the modification(s) in most cases. In all but one case, unambiguous modification site determination was achieved with MAD. MAD produced 15–20% better sequence coverage than CID and ETD for both the 2+ and 3+ charge states and very different fragmentation products indicating that the mechanism of fragmentation in MAD is unique and complementary to CID and ETD. Copyright © 2012 John Wiley & Sons, Ltd.     

Study of the dissociation of a charge-reduced phosphopeptide formed by electron transfer from an alkali metal target

Doubly protonated phosphopeptide (YGGMHRQET(p)VDC) ions obtained by electrospray ionization were collided with Xe and Cs targets to give singly and doubly charged positive ions via collision-induced dissociation (CID). The resulting ions were analyzed and detected by using an electrostatic analyzer (ESA). Whereas doubly charged fragment ions resulting from collisionally activated dissociation (CAD) were dominant in the CID spectrum with the Xe target, singly charged fragment ions resulting from electron transfer dissociation (ETD) were dominant in the CID spectrum with the Cs target. The most intense peak resulting from ETD was estimated to be associated with the charge-reduced ion with H2 lost from the precursor. Five c-type fragment ions with amino acid residues detached consecutively from the C-terminal were clearly observed without a loss of the phosphate group. These ions must be formed by N--Calpha bond cleavage, in a manner similar to the cases of electron capture dissociation (ECD) and ETD from negative ions. Although the accuracy in m/z of the CID spectra was about +/-1 Th because of the mass analysis using the ESA, it is supposed from the m/z values of the c-type ions that these ions were accompanied by the loss of a hydrogen atom. Four z-type (or y--NH3, or y--H2O) ions analogously detached consecutively from the N-terminal were also observed. The fragmentation processes took place within the time scale of 4.5 micros in the high-energy collision. The present results demonstrated that high-energy ETD with the alkali metal target allowed determination of the position of phosphorylation and the amino acid sequence of post-translational peptides.     

Fragmentation behavior of Amadori‐peptides obtained by non‐enzymatic glycosylation of lysine residues with ADP‐ribose in tandem mass spectrometry

Mono‐ and poly‐adenosine diphosphate (ADP)‐ribosylation are common post‐translational modifications incorporated by sequence‐specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non‐enzymatic ADP‐ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori‐compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix‐assisted laser desorption/ionization (MALDI)‐MS, the ADP‐ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in‐source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)‐MS. The same fragmentation site also dominated the MALDI‐PSD (post‐source decay) and ESI‐CID (collision‐induced dissociation) mass spectra. The remaining phospho‐ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b‐ and y‐ion series. Cleavage of the ADP‐ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor‐ion scans (m/z 348.08), and thereby permits the identification of ADP‐ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP‐ribosyl group was stable, providing ADP‐ribosylated c‐ and z‐ions, and thus allowing reliable sequence analyses. Copyright © 2010 John Wiley & Sons, Ltd.     

Tandem mass spectrometry of multiply phosphorylated forms of a 'histidine-tag' derived from a recombinant protein kinase expressed in bacteria

When a histidine-tagged form of the protein kinase Aurora-2 was expressed in Escherichia coli, the purified product carried four to nine phosphate groups, although many fewer were expected. The amino-terminal tag had the sequence GSSHHHHHHSSGLVPRGSHMK-. Tryptic digestion of the product followed by analysis by liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) showed that phosphorylation could occur on the five serine residues of the tag. Mono-, bis-, tris-, tetra- and pentaphosphorylated forms of the tag were detected, and their behavior in MS/MS was studied using a quadrupole/time-of-flight mass spectrometer. The MS/MS spectra were dominated by the products of neutral loss events (in 98 Da increments, each equivalent to loss of H3PO4), but sufficient b- and y-type sequence ions were detected to allow the locations of the phosphates to be specified in some cases. The assignment of phosphorylation sites for incompletely phosphorylated forms of the tag peptide was challenging, but it appeared that Ser-10 and Ser-11 of the tag were more likely to be phosphorylated than Ser-2 and Ser-3.     

Effects of stepwise excitation on fragmentation of gas phase ions: An especially striking case

Dramatically different fragmentation patterns are obtained for 4-ethyl-2,6,7-trioxa-1-phosphabicyclo-[2.2.2]octane-1-oxide upon electron ionization (EI) and for the corresponding molecular ion on collision-induced dissociation (CID). Two reasons for this behaviour have been discovered. (i) Irreversible multistep isomerization of the molecular ions occurs prior to collisional activation in mass spectrometry/mass spectrometry (MS/MS). Isomerization reactions have been characterized by isotopic labelling and by examining structures of relevant unlabelled and labelled fragment ions by MS/MS. The extent of isomerization can be controlled by varying the amount of internal energy of the molecular ions. This has been done by changing the number of thermalizing collisions which the ions undergo with neutral molecules in the ion source. (ii) When multiple collisions are used to dissociate the molecular ions, the initially stable fragmentation products undergo extensive further decomposition. As a result, abundant phosphorus-containing fragment ions are obtained for the bicyclic phosphate in high-pressure CID, whereas electron ionization leads to predominant hydrocarbon ions. A minor change in the structure of this molecule has major effects on the fragmentation behaviour: high- and low-energy collisional activation spectra of the molecular ion of the corresponding phosphite are identical with the 12 e V EI mass spectrum of the neutral.     

Electron capture dissociation of singly and multiply phosphorylated peptides

Analysis of phosphotyrosine and phosphoserine containing peptides by nano-electrospray Fourier transform ion cyclotron resonance (FTICR) mass spectrometry established electron capture dissociation (ECD) as a viable method for phosphopeptide sequencing. In general, ECD spectra of synthetic and native phosphopeptides appeared less complex than conventional collision activated dissociation (CAD) mass spectra of these species. ECD of multiply protonated phosphopeptide ions generated mainly c- and z(.)-type peptide fragment ion series. No loss of water, phosphate groups or phosphoric acid from intact phosphopeptide ions nor from the c and z(.) fragment ion products was observed in the ECD spectra. ECD enabled complete or near-complete amino acid sequencing of phosphopeptides for the assignment of up to four phosphorylation sites in peptides in the mass range 1400 to 3500 Da. Nano-scale Fe(III)-affinity chromatography combined with nano-electrospray FTMS/ECD facilitated phosphopeptide analysis and amino acid sequencing from crude proteolytic peptide mixtures.     

The use of ECD/ETD to identify the site of electrostatic interaction in noncovalent complexes

Electrostatic interactions play an important role in the formation of noncovalent complexes. Our previous work has highlighted the role of certain amino acid residues, such as arginine, glutamate, aspartate, and phosphorylated/sulfated residues, in the formation of salt bridges resulting in noncovalent complexes between peptides. Tandem mass spectrometry (MS) studies of these complexes using collision-induced dissociation (CID) have provided information on their relative stability. However, product-ion spectra produced by CID have been unable to assign specifically the site of interaction for the complex. In this work, tandem MS experiments were conducted on noncovalent complexes using both electron capture dissociation (ECD) and electron-transfer dissociation (ETD). The resulting spectra were dominated by intramolecular fragments of the complex with the electrostatic interaction site intact. Based upon these data, we were able to assign the binding site for the peptides forming the noncovalent complex.     

Effects of Electron-Transfer Coupled with Collision-Induced Dissociation (ET/CID) on Doubly Charged Peptides and Phosphopeptides

Electron-transfer dissociation (ETD) is a useful peptide fragmentation technique that can be applied to investigate post-translational modifications (PTMs), the sequencing of highly hydrophilic peptides, and the identification of large peptides and even intact proteins. In contrast to traditional fragmentation methods, such as collision-induced dissociation (CID), ETD produces c- and z^·-type product ions by randomly cleaving the N–Cα bonds. The disappointing fragmentation efficiency of ETD for doubly charged peptides and phosphopeptide ions has been improved by ETcaD (supplemental activation). However, the ETD data derived from most database search algorithms yield low confidence scores due to the presence of unreacted precursors and charge-reduced ions within MS/MS spectra. In this work, we demonstrate that eight out of ten standard doubly charged peptides and phosphopeptides can be effortlessly identified by electron-transfer coupled with collision-induced dissociation (ET/CID) using the SEQUEST algorithm without further spectral processing. ET/CID was performed with the further dissociation of the charge-reduced ions isolated from ETD ion/ion reactions. ET/CID had high fragmentation efficiency, which elevated the confidence scores of doubly charged peptide and phosphospeptide sequencing. ET/CID was found to be an effective fragmentation strategy in “bottom-up” proteomic analysis.     

Pseudo-MS3 in a MALDI orthogonal quadrupole-time of flight mass spectrometer

Both the matrix selected and the laser fluence play important roles in MALDI-quadrupole/time of flight (QqTOF) fragmentation processes. "Hot" matrices, such as alpha-cyano4-hydroxycinnamic acid (HCCA), can increase fragmentation in MS spectra. Higher laser fluence also increases fragmentation. Typical peptide fragment ions observed in the QqTOF are a, b, and y ion series, which resemble low-energy CID product ions. This fragmentation may occur in the high-pressure region before the first mass-analyzing quadrupole. Fragment ions can be selected by the first quadrupole (Q1), and further sequenced by conventional MS/MS. This allows pseudo-MS3 experiments to be performed. For peptides of higher molecular weight, pseudo-MS3 can extend the mass range beyond what is usually accessible for sequencing, by allowing one to sequence a fragment ion of lower molecular weight instead of the full-length peptide. Peptides that predominantly show a single product ion after MS/MS yield improved sequence information when this technique is applied. This method was applied to the analysis of an in vitro phosphorylated peptide, where the intact enzymatically-generated peptide showed poor dissociation via MS/MS. Sequencing a fragment ion from the phosphopeptide enabled the phosphorylation site to be unambiguously determined.     

UV photodissociation of phospho-seryl-containing peptides: laser stabilization of the phospho-seryl bond with multistage mass spectrometry

Protonated precursor ions of phosphorylated peptides containing a tyrosyl residue have been subjected to UV laser-induced dissociation (LID) at a wavelength of 220 nm and to collision-induced dissociation (CID) in an ion trap. As expected, neutral loss of the phosphate group is one of the predominant fragmentation channels during CID together with H2O elimination. In contrast, LID leads mainly to the homolytic cleavage of the tyrosyl side chain and a restrained loss of the phosphate group. Interestingly, the intensity of the dephosphorylated fragment ion is greatly minimized when CID is carried out next on the radical precursor ion of the singly and doubly charged species.     

De novo peptide sequencing by tandem MS using complementary CID and electron transfer dissociation

De novo sequencing of peptides using tandem MS is difficult due to missing fragment ions in the spectra commonly obtained after CID of peptide precursor ions. Complementing CID spectra with spectra obtained in an ion‐trap mass spectrometer upon electron transfer dissociation (ETD) significantly increases the sequence coverage with diagnostic ions. In the de novo sequencing algorithm CompNovo presented here, a divide‐and‐conquer approach was combined with an efficient mass decomposition algorithm to exploit the complementary information contained in CID and ETD spectra. After optimizing the parameters for the algorithm on a well‐defined training data set obtained for peptides from nine known proteins, the CompNovo algorithm was applied to the de novo sequencing of peptides derived from a whole protein extract of Sorangium cellulosum bacteria. To 2406 pairs of CID and ETD spectra contained in this data set, 675 fully correct sequences were assigned, which represent a success rate of 28.1%. It is shown that the CompNovo algorithm yields significantly improved sequencing accuracy as compared with published approaches using only CID spectra or combined CID and ETD spectra.     

Determination of pyridoxal‐5′‐phosphate (PLP)‐bonding sites in proteins: a peptide mass fingerprinting approach based on diagnostic tandem mass spectral features of PLP‐modified peptides

Peptides modified by pyridoxal‐5′‐phosphate (PLP), linked to a lysine residue via reductive amination, exhibit distinct spectral characteristics in the collision‐induced dissociation (CID) tandem mass (MS/MS) spectra that are described here. The MS/MS spectra typically display two dominant peaks whose m/z values correspond to neutral losses of [H₃PO₄] (?98 Da) and the PLP moiety as [C₈H₁₀NO₅P] (?231 Da) from the precursor peptide ion, respectively. Few other peaks are observed. Recognition of this distinct fragmentation behavior is imperative since determining sequences and sites of modifications relies on the formation of amide backbone cleavage products for subsequent interpretation via proteome database searching. Additionally, PLP‐modified peptides exhibit suppressed precursor ionization efficiency which diminishes their detection in complex mixtures. Presented here is a protocol which describes an enrichment strategy for PLP‐modified peptides combined with neutral loss screening and peptide mass fingerprinting to map the PLP‐bonding site in a known PLP‐dependent protein. This approach represents an efficient alternative to site‐directed mutagenesis which has been the traditional method used for PLP‐bonding site localization in proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Mechanistic insights into intramolecular phosphate group transfer during collision induced dissociation of phosphopeptides

We report on the rearrangement chemistry of model phosphorylated peptides during collision‐induced dissociation (CID), where intramolecular phosphate group transfers are observed from donor to acceptor residues. Such “scrambling” could result in inaccurate modification localization, potentially leading to misidentifications. Systematic studies presented herein provide mechanistic insights for the unusually high phosphate group rearrangements presented some time ago by Reid and coworkers (Proteomics 2013, 13 [6], 964‐973). It is postulated here that a basic residue like histidine can play a key role in mediating the phosphate group transfer by deprotonating the serine acceptor site. The proposed mechanism is consistent with the observation that fast collisional activation by collision‐cell CID and higher‐energy collisional dissociation (HCD) can shut down rearrangement chemistry. Additionally, the rearrangement chemistry is highly dependent on the charge state of the peptide, mirroring previous studies that less rearrangement is observed under mobile proton conditions.     

Analysis of non-enzymatically glycated peptides: neutral-loss-triggered MS(3) versus multi-stage activation tandem mass spectrometry

Non-enzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. While electron transfer dissociation (ETD) has been shown to outperform collision-induced dissociation (CID) in sequencing glycated peptides by tandem mass spectrometry, ETD instrumentation is not yet widely available and often suffers from significantly lower sensitivity than CID. In this study, we evaluated different advanced CID techniques (i.e., neutral-loss-triggered MS(3) and multi-stage activation) during liquid chromatography/multi-stage mass spectrometric (LC/MS(n)) analyses of Amadori-modified peptides enriched from human serum glycated in vitro. During neutral-loss-triggered MS(3) experiments, MS(3) scans triggered by neutral losses of 3 H(2)O or 3 H(2)O + HCHO produced similar results in terms of glycated peptide identifications. However, neutral losses of 3 H(2)O resulted in significantly more glycated peptide identifications during multi-stage activation experiments. Overall, the multi-stage activation approach produced more glycated peptide identifications, while the neutral-loss-triggered MS(3) approach resulted in much higher specificity. Both techniques are viable alternatives to ETD for identifying glycated peptides. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Fragmentation of phosphopeptides by atmospheric pressure MALDI and ESI/ion trap mass spectrometry

An investigation of phosphate loss from phosphopeptide ions was conducted, using both atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) and electrospray ionization (ESI) coupled to an ion trap mass spectrometer (ITMS). These experiments were carried out on a number of phosphorylated peptides in order to investigate gas phase dephosphorylation patterns associated with phosphoserine, phosphothreonine, and phosphotyrosine residues. In particular, we explored the fragmentation patterns of phosphotyrosine containing peptides, which experience a loss of 98 Da under collision induced dissociation (CID) conditions in the ITMS. The loss of 98 Da is unexpected for phosphotyrosine, given the structure of its side chain. The fragmentation of phosphoserine and phosphothreonine containing peptides was also investigated. While phosphoserine and phosphothreonine residues undergo a loss of 98 Da under CID conditions regardless of peptide amino acid composition, phosphate loss from phosphotyrosine residues seems to be dependent on the presence of arginine or lysine residues in the peptide sequence.     

Collision-induced dissociation of glycero phospholipids using electrospray ion-trap mass spectrometry.

Characterisation of phospholipids was achieved using collision-induced dissociation (CID) with an ion-trap mass spectrometer. The product ions were compared with those obtained with a triple quadrupole mass spectrometer. In the negative ion mode the product ions were mainly sn-1 and sn-2 lyso-phospholipids with neutral loss of ketene in combination with neutral loss of the polar head group. Less abundant product ions were sn-1 and sn-2 carboxylate anions. CID using a triple quadrupole mass spectrometer, however, gave primarily the sn-1 and sn-2 carboxylate anions together with lyso-phosphatidic acid with neutral loss of water. For the ion trap a charge-remote-type mechanism is proposed for formation of the lyso-phospholipid product ions by loss of alpha-hydrogen on the fatty acid moiety, electron rearrangement and neutral loss of ketene. A second mechanism involves nucleophilic attack of the phosphate oxygen on the sn-1 and sn-2 glycerol backbone to form carboxylate anions with neutral loss of cyclo lyso-phospholipids. CID (MS(3) and MS(4)) of the lyso-phospholipids using the ion-trap gave the same carboxylate anions as those obtained with a triple quadrupole instrument where multiple collisions in the collision cell are expected to occur. The data demonstrate that phospholipid species determination can be performed by using LC/MS(n) with an ion-trap mass spectrometer with detection of the lyso-phospholipid anions. The ion-trap showed no loss in sensitivity in full scan MS(n) compared to multiple reaction monitoring data acquisition. In combination with on-line liquid chromatography this feature makes the ion-trap useful in the scanning modes for rapid screening of low concentrations of phospholipid species in biological samples as recently described (Uran S, Larsen A, Jacobsen PB, Skotland T. J. Chromatogr. B 2001; 758: 265).     

Comparison of the activation time effects and the internal energy distributions for the CID,PQD and HCD excitation modes

Reproducibility among different types of excitation modes is a major bottleneck in the field of tandem mass spectrometry library development in metabolomics. In this study, we specifically evaluated the influence of collision voltage and activation time parameters on tandem mass spectrometry spectra for various excitation modes [collision‐induced dissociation (CID), pulsed Q dissociation (PQD) and higher‐energy collision dissociation (HCD)] of Orbitrap‐based instruments. For this purpose, internal energy deposition was probed using an approach based on Rice–Rampserger–Kassel–Marcus modeling with three thermometer compounds of different degree of freedom (69, 228 and 420) and a thermal model. This model treats consecutively the activation and decomposition steps, and the survival precursor ion populations are characterized by truncated Maxwell–Boltzmann internal energy distributions. This study demonstrates that the activation time has a significant impact on MS/MS spectra using the CID and PQD modes. The proposed model seems suitable to describe the multiple collision regime in the PQD and HCD modes. Linear relationships between mean internal energy and collision voltage are shown for the latter modes and the three thermometer molecules. These results suggest that a calibration based on the collision voltage should provide reproducible for PQD, HCD to be compared with CID in tandem in space instruments. However, an important signal loss is observed in PQD excitation mode whatever the mass of the studied compounds, which may affect not only parent ions but also fragment ions depending on the fragmentation parameters. A calibration approach for the CID mode based on the variation of activation time parameter is more appropriate than one based on collision voltage. In fact, the activation time parameter in CID induces a modification of the collisional regime and thus helps control the orientation of the fragmentation pathways (competitive or consecutive dissociations). Copyright © 2014 John Wiley & Sons, Ltd.     

Electron Transfer Dissociation of Milk Oligosaccharides

For structural identification of glycans, the classic collision-induced dissociation (CID) spectra are dominated by product ions that derived from glycosidic cleavages, which provide only sequence information. The peaks from cross-ring fragmentation are often absent or have very low abundances in such spectra. Electron transfer dissociation (ETD) is being applied to structural identification of carbohydrates for the first time, and results in some new and detailed information for glycan structural studies. A series of linear milk sugars was analyzed by a variety of fragmentation techniques such as MS/MS by CID and ETD, and MS(3) by sequential CID/CID, CID/ETD, and ETD/CID. In CID spectra, the detected peaks were mainly generated via glycosidic cleavages. By comparison, ETD generated various types of abundant cross-ring cleavage ions. These complementary cross-ring cleavages clarified the different linkage types and branching patterns of the representative milk sugar samples. The utilization of different MS(3) techniques made it possible to verify initial assignments and to detect the presence of multiple components in isobaric peaks. Fragment ion structures and pathways could be proposed to facilitate the interpretation of carbohydrate ETD spectra, and the main mechanisms were investigated. ETD should contribute substantially to confident structural analysis of a wide variety of oligosaccharides.