Top-down sequencing of O-glycoproteins by in-source decay matrix-assisted laser desorption ionization mass spectrometry for glycosylation site analysis

The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17-18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154.     

Multistage and tandem mass spectrometry of glycosylated triterpenoid saponins isolated from Bacopa monnieri: comparison of the information content provided by different techniques

Whereas all state-of-the-art techniques in mass spectrometry (MS) have been extensively applied to oligosaccharides derived from glycoproteins, less effort has been devoted to the analysis of smaller glycoconjugates. In the present study, the application of a variety of MS techniques for the analysis of two dammarane-type triterpenoid saponins isolated from B. monnieri is reported. The structural information provided by ESI-ion trap (IT)-, AP-MALDI-IT-, and MALDI-IT/reflectron time-of-flight (RTOF)-MS, all utilizing low-energy collision-induced dissociation (CID), and MALDI-TOF/RTOF-MS, facilitating postsource decay and high-energy CID analysis, was compared. The applied desorption/ionization technique does not influence the fragmentation of identical precursor ions in low-energy CID. All three fragmentation techniques clearly yield the sequence and branching of the glycan moiety as well as the molecular mass of the intact aglycon. Cross-ring cleavage of the branching sugar, which gives some information about the sugar linkages, was mainly observed in low-energy CID. High-energy CID, on the other hand, yielded some additional diagnostic fragment ions from the aglycon moiety. Internal cleavage ions are formed by alternative mechanisms in high-energy CID and are assumed to be diagnostic for some linkages. However, none of the applied MS techniques facilitates the identification of those saponins that differ only by their aglycon moiety (i.e., jujubogenin or pseudojujubogenin).     

Improvement of the MS/MS fragment ion coverage of acidic residue-containing peptides by amidation with 15N-substituted amine

Tandem mass spectrometry (MS/MS) is a powerful tool for peptide sequencing and characterization. However, the selective cleavage at acidic residues, aspartic acid, and glutamic acid prevents the generation of enough product ions to elucidate the entire sequence. We attempted to solve the problem by converting the residues into the corresponding amides, asparagine and glutamine. The amidation suppressed the cleavage at the converted residues, and the product ions derived from dissociation at other sites became abundant. Incorporation of nitrogen isotope (15)N in the amine constituent for amidation minimized the mass change from -0.984 016 to +0.013 019, allowing easy discrimination of acidic and amide residues in the original sequences by MS/MS database search. In addition, the amidated and unchanged peptides had the same nominal mass, even when the transformation was incomplete, which was approximately 70% in the current condition. The unmodified acidic residues remaining were rather useful to give marker fragments by the dominant dissociation. These results demonstrate that (15)N-amidation is effective in improving the performance of MS/MS to elucidate amino acid sequences of peptides.     

Anionic adducts of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The formation and decomposition (postsource decay, PSD) of anionic adducts in negative ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry have been studied. Chloride, a small inorganic anion, has been found to form stable anionic adducts with a variety of neutral oligosaccharides that can survive the MALDI process to give readily identifiable signals (with characteristic isotope patterns) allowing subpicomole detection in the best cases. The matrixes that can aid the formation of chloride adducts of oligosaccharides have gas-phase acidities lower than or close to that of HCl (1373 kJ/mol). In PSD experiments, precursor chloride adducts of oligosaccharides yield fragment ions that retain the charge on the sugar molecule rather than solely forming Cl-, and these fragments can provide structurally informative product ions. In negative ion MALDI, highly acidic oligosaccharides do not form adducts with chloride anions, but mildly acidic saccharides (e.g., containing a carboxylic acid group) form both deprotonated molecules and chloride adducts, and each may provide structural information concerning the oligosaccharide upon decomposition.     

High ion yields of carbohydrates from frozen solution by UV-MALDI

An aqueous acetonitrile solution containing oligosaccharides (maltopentaose and polysaccharides) and a matrix (2,5-dihydroxybenzoic acid) was frozen at 100 K for mass analysis using ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI). Compared with conventional UV-MALDI (i.e., using a dry analyte/matrix mixture), a frozen solution generates more oligosaccharide ions and less fragments from postsource decay. Furthermore, the ion signal is long-lasting, and the analyte distribution features enhanced homogeneity. The ion generation efficiency for this procedure is 20-30 times greater than that for a conventional dried mixture. Interestingly, the percentages for maltopentaose fragmentation from postsource decay for the frozen samples are close to zero (<2%), as compared with the 17% and 40% values found for dried samples at low and high laser fluences, respectively. Comparisons with other UV matrixes (α-cyano-4-hydroxycinnamic acid and sinapinic acid) and ionic liquids (2,5-dihydroxybenzoic acid + pyridine and α-cyano-4-hydroxycinnamic acid + butylamine) were investigated, and possible mechanisms are discussed.     

Localization of the O-glycosylated sites in peptides by fixed-charge derivatization with a phosphonium group

The present study demonstrates that matrix-assisted laser desorption ionization/postsource decay (MALDI/PSD) analysis of the molecular cation of glycopeptides derivatized at their amino terminus with a phosphonium group cleaves peptide backbone without removing the glycan. The predictable a-type fragment ions retain the glycan moiety, enabling unambiguous localization of O-glycans on the peptide chain. In contrast, collision-activated dissociation tandem mass spectrometry analysis carried out on the doubly charged protonated phosphonium cation results in the predominant loss of the sugar moiety from the peptide. This result supports the previously proposed charge-induced fragmentation mechanism of the sugar-peptide bond. MALDI/PSD analysis of glycopeptides converted to their acetyl phosphonium derivatives is an effective alternative to electron capture dissociation, as illustrated by the positioning of up to three GalNac residues along the full tandem repeat peptide sequence derived from the MUC 5AC mucin.     

Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry

The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique described to date has proven to be a convenient and rapid method for identification and characterizations of proteins. However, the general MALDI-TOF MS analysis of complex carbohydrates and glycopeptides still entails special consideration of ionization and the fragmentation characteristics of labile carbohydrate moieties. In this study, an efficient and practical method we termed the matrix-dependent selective fragmentation (MDSF) technique of MALDI-TOF/TOF MS, which allows highly sensitive and reliable fragmentation of oligosaccharides and N-glycopeptides. Results from application of the MDSF technique to TOF/TOF MS analysis demonstrated that in comparison to the conventional postsource decay up to 170 times more sensitive product ion peaks could be obtained. It was also suggested that MDSF generates desired structural information based on the controlled cleavage of the singly charged precursor ion with different electronic excited states made by this method. Ideal product ion peaks observed by MDSF in TOF/TOF MS facilitated structural characterization of complex oligosaccharide derivatives including unstable Neu5Ac and Fuc residues and N-glycopeptides.     

A CE-MALDI interface based on the use of prestructured sample supports

We have developed an off-line coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) based on CE fraction collection onto prestructured MALDI sample supports. Analyte carryover and detection sensitivity were investigated using a standard peptide mixture. Low femtomole amounts were detected, and no noticeable carryover was discovered. The performance of the method was evaluated with a mixture of tryptic digests of proteins from a human fetal brain cDNA expression library. The total number of identified peptides was increased from 47 to 211 when the CE-MALDI interface was used compared to direct MALDI-MS analysis. Sequence coverage with CE-MALDI was in the 25-60% range for the different proteins, corresponding to an increase of 1.3-4.9 times relative to that obtained with MALDI-MS of the crude mixture. Fractionation of sample components also facilitated protein identification by MALDI postsource decay analysis. Our initial results suggest this CE-MALDI interface can be used for the analysis of complex peptide mixtures isolated from biological tissues.     

Enhancement of MALDI-MS spectra of C-terminal peptides by the modification of proteins via an active ester generated in situ from an oxazolone

For selective C-terminal derivatization of peptides and proteins, we have devised a method for activating the C-terminal carboxyl group by extending the oxazolone chemistry. A mixture of formic acid and acetic anhydride was found to be effective for the formation of an oxazolone, which was converted to an active ester in situ in the presence of a phenol or an N-hydroxide. In particular, the resulting active ester with pentafluorophenol facilitated the subsequent reaction with an amine and the hydrazine derivative to yield the C-terminal amide and hydrazide, respectively. The peptides thus coupled with arginine methyl ester or 2-hydrazino-2-imidazoline containing the guanidino moiety exhibited the positive-ion peaks in matrix-assisted laser desorption/ionization (MALDI) mass spectra with appreciably enhanced intensities. As expected from the reaction mechanism, the carboxyl groups of aspartic and glutamic acid residues were not modified, while the amino groups that could react with the activated peptides were concomitantly protected by formylation. The MALDI peaks corresponding to the C-terminal peptide fragments of proteins were specifically enhanced, discriminating against those from internal peptides that were not tagged with a positive charge. In favorable cases, the C-terminal peptide fragments were clearly discerned by MALDI-MS after chymotryptic digestion and were identified by their MALDI postsource decay analysis. Based on these results, we suggest a method for C-terminal sequencing of a protein.     

Enabling MALDI-FTICR-MS/MS for high-performance proteomics through combination of infrared and collisional activation

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a central tool for proteomic analysis, yet the singly protonated tryptic peptide ions produced by MALDI are significantly more difficult to dissociate for tandem mass spectrometry (MS/MS) than the corresponding multiply protonated ions. In order to overcome this limitation, current proteomic approaches using MALDI-MS/MS involve high-energy collision-induced dissociation (CID). Unfortunately, the use of high-energy CID complicates product ion spectra with a significant proportion of irrelevant fragments while also reducing mass accuracy and mass resolution. In order to address the lack of a high-resolution, high mass accuracy MALDI-MS/MS platform for proteomics, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and a recently developed MS/MS technique termed CIRCA (for combination of infrared and collisional activation) have been applied to proteomic analysis. Here, CIRCA is shown to be suitable for dissociating singly protonated tryptic peptides, providing greater sequence coverage than either CID or infrared multiphoton dissociation (IRMPD) alone. Furthermore, the CIRCA fragmentation spectra are of sufficient quality to allow protein identification based on the MS/MS spectra alone or in concert with the peptide mass fingerprint (PMF). This is accomplished without compromising mass accuracy or mass resolution. As a result, CIRCA serves to enable MALDI-FTICR-MS/MS for high-performance proteomics experiments.     

Identification of 4-hydroxy-2-nonenal-modified peptides within unfractionated digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is generated as a consequence of oxidative stress and can readily react with nucleophilic sites of proteins (e.g., histidine residues), mainly via a Michael addition. The formation of such lipid-protein conjugates can alter protein properties and biological functions, thus leading to highly deleterious effects. The present work describes a rapid (very limited sample preparation) and sensitive (low-femtomole range) procedure to identify HNE-modified peptides (Michael adducts) within unfractionated tryptic digests. The protocol involves the formation of dinitrophenylhydrazones of the Michael adducts, when using 2,4-dinitrophenylhydrazine as reactive matrix, followed by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives present high desorption/ionization yield and can thus be preferentially detected compared to unmodified peptides. The MALDI mass spectrum obtained is therefore drastically different from the one obtained with the classical 4-hydroxy-alpha-cyanocinnamic acid matrix. Moreover, the presence of HNE, or more generally speaking carbonylated peptides, could be highlighted by 180 mass units differences (corresponding to the dinitrophenylhydrazone moiety) between these two MALDI mass spectra. Further information (e.g., localization/identification of the modified residues, peptide sequences) could be obtained by performing MALDI postsource decay (or electrospray) MS/MS experiments on the ions of interest.     

In situ sequencing of peptides from biological tissues and single cells using MALDI-PSD/CID analysis

The ability to directly sequence peptides from biological cells using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with postsource decay (PSD) and collision-induced dissociation (CID) fragment ion mass analysis is explored. Three different sample preparation methods are described for sequencing peptides in tissue samples and in single neurons from the invertebrate model Aplysia californica. To characterize peptides from the atrial gland, MALDI-PSD/CID is applied directly to a tissue blot covered with the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). The resulting fragment ions combined with database searching confirm the structure of several novel peptides encoded by egg-laying hormone genes. Moreover, MS profiling of a single unidentified neuron detects peptides with molecular weights of myomodulins C and E; this assignment is confirmed using MALDI-PSD with the matrix 2,5-dihydroxybenzoic acid (DHB). DHB does not always provide adequate fragmentation for PSD experiments; therefore, a unique dual-matrix sampling method, employing both DHB and CHCA, is developed to directly sequence a decapeptide from a single cerebral ganglion B cell. Mass accuracy of fragment ions from cellular samples is typical for the instrument employed and is not deleteriously affected by the morphology and complexity of the samples.     

In-source and postsource decay in negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of neutral oligosaccharides

Cross-ring cleavage ions produced by in-source decay (ISD), as well as deprotonated molecular ions [M - H]-, are invariably observed in negative-ion linear-mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectra of neutral oligosaccharides with 9H-pyrido[3,4-b]indole (norharman) as a matrix. The patterns of ISD ions depend on the oligosaccharide linkage type; thus, these ions are potentially useful in linkage analysis. In postsource decay (PSD) spectra from chlorinated molecular ions [M + Cl]-, all PSD ions are observed in the deprotonated form, although no deprotonated molecular ions are detected. In oligosaccharides having an alditol at the reducing end, deprotonated molecular ions [M - H]- are clearly seen in linear-mode mass spectra and survive in the PSD measurements. These results indicate that the deprotonation process drives ISD and PSD of oligosaccharides and that keto-enol tautomerization at the reducing terminal promotes ISD and PSD processes.     

Neutral loss analysis in MALDI-MS using an ion-gate scanning mode.

Matrix-assisted laser desorption/ionization reflector time-of-flight (MALDI-reTOF) and electrospray ionization (ESI) mass spectrometry (MS) have become essential tools for the characterization of peptides and proteins. Whereas ESI in combination with a triple quadrupole analyzer allows product ion, precursor ion, and neutral loss analyses, MALDI-reTOF instruments can only be used to record product ion spectra based on the in-source or postsource decay (PSD). We describe a new method to perform neutral loss analyses in MALDI-reTOF instruments in a manner that identifies posttranslationally modified peptides and furthermore retrieves sequence information from peptides. The method is based on the selection of ions in a small time interval to record only signals within the corresponding mass interval. By stepping the time interval through the complete mass range, we obtained a spectrum of stable ions by combining the signals of all individually recorded time intervals. This method furthermore permits PSD fragment ions to be identified, since they reach the detector earlier than the stable ions transmitted in the chosen time interval. The neutral loss analysis were calculated by correlating the PSD fragment ions to the corresponding parent ion detected in this time interval. Moreover, this MALDI-MS mode increased the number of detectable signals in complex peptide mixtures and the signal-to-noise ratio.     

Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry

The direct profiling of proteins present in tissue sections for several organs of the mouse has been accomplished using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Fresh tissue was sectioned and blotted on a conductive polyethylene membrane. The dried membrane blot was coated with matrix, typically sinapinic acid, and directly analyzed in the mass spectrometer. Generally, well over 100 peptide/protein signals in the 2000-30,000 Da range were observed, with 30-50 having relatively high signal intensities. Analysis of different areas of the same tissue gave remarkably similar mass spectra with greater than 90% homology. However, different parts of a segmented tissue, such as the proximal, intermediate, and distal colon, gave some unique protein signals. After treatment of the tissue blot with protease and subsequent MALDI MS analysis using postsource decay methods for peptide sequencing, some of the proteins were identified. The unique protein profiles measured from these tissue blots also showed differences from strain to strain of the mouse, with genetically similar strains having very similar patterns.     

Discrimination of neolacto-series gangliosides with alpha2-3- and alpha2-6-linked N-acetylneuraminic acid by nanoelectrospray ionization low-energy collision-induced dissociation tandem quadrupole TOF MS

A combined strategy of thin-layer chromatography immunostaining and negative ion nanoelectrospray low-energy CID mass spectrometry was established for the differentiation of isomeric alpha2-3 and alpha2-6 sialylated neolacto-series monosialogangliosides from human granulocytes. The gangliosides investigated differed in the ceramide moiety by substitution with C16:0 or C24:1 fatty acid and in their oligosaccharide chains due to nLc4 and nLc6 core structures. With respect to the type of sialylation, the homogeneity of the HPLC-purified ganglioside fractions was verified by use of specific anti-Neu5Acalpha2-3Galbeta1-4GlcNAc-R and anti-Neu5Acalpha2-6Galbeta1-4GlcNAc-R antibodies. A clear-cut series of fragment ions for both types of isomeric gangliosides, carrying alpha2-3- and alpha2-6-linked neuraminic acid, respectively, was obtained by low-energy CID. Additionally, a characteristic ring cleavage was detected exclusively in all species with Neu5Acalpha2-6Galbeta1-4GlcNAc terminus, regardless of ceramide fatty acid and oligosaccharide chain lengths. The diagnostic (0,2)X(4/6) ions, generated by ring cleavage of an alpha2-6-linked neuraminic acid are accompanied by a simultaneous decrease of the corresponding Y(4)/Y(6) ions. These results suggest the unequivocal discrimination of individual alpha2-3- and alpha2-6-sialylated neolacto-series monosialogangliosides by distinct fragmentation patterns in low-energy CID tandem MS.     

Negative-ion electrospray mass spectrometry of neutral underivatized oligosaccharides

Negative-ion electrospray mass spectrometry (ES-MS) with collision-induced dissociation (CID) and MS/MS scanning on a quadrupole-orthoganal time-of-flight instrument provide a sensitive means for structural analysis of neutral underivatized oligosaccharides. Molecular mass information can be readily obtained from the dominant [M - H]- ions in the ES mass spectrum formed with subnanomole amounts of oligosaccharides, and similar sensitivity is available from CID-MS/MS to give structural details. The CID spectra of 14 oligosaccharides demonstrated that sequence and partial linkage information can be derived and isomeric structures can be differentiated. Series of C-type fragment ions give sequence information while the double glycosidic D-type cleavage of a 3-linked GlcNAc or Glc and the saccharide ring fragmentation of the 0,2A-type from 4-linked GlcNAc or Glc can provide partial linkage information. The distinctive D- and A-cleavages are important for differentiation of oligosaccharide type 1 and type 2 chains and to define the blood group H, Le(a), Le(x), Le(b), and Le(y) determinants carried by their fucosylated analogues.     

Enhanced ionization of phosphorylated peptides during MALDI TOF mass spectrometry

Although alpha-cyano-4-hydroxycinnamic acid functions as an excellent matrix for the analysis of most peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry, the ionization of phosphorylated peptides is usually suppressed by nonphosphorylated peptides. As an alternative matrix, 2',4',6'-trihydroxyacetophenone (THAP) with diammonium citrate was found to overcome this problem for the MALDI TOF mass spectrometric analysis of proteolytic digests of phosphorylated proteins. Specifically, the abundances of phosphorylated peptides in tryptic digests of bovine beta-casein and protein kinase C (PKC)-treated mouse cardiac troponin I were enhanced more than 10-fold using THAP during positive ion MALDI TOF mass spectrometry. The protonated molecules of phosphorylated peptides were sufficiently abundant that postsource decay TOF mass spectrometry was used to confirm the number of phosphate groups in each peptide. Finally, tryptic digestion followed by analysis using MALDI TOF mass spectrometry with THAP as the matrix facilitated the identification of a unique phosphorylation site in PKC-treated troponin I.     

Imaging MALDI mass spectrometry using an oscillating capillary nebulizer matrix coating system and its application to analysis of lipids in brain from a mouse model of Tay-Sachs/Sandhoff disease

The quality of tissue imaging by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) depends on the effectiveness of the matrix deposition, especially for lipids that may dissolve in the solvent used for the matrix application. This article describes the use of an oscillating capillary nebulizer (OCN) to spray small droplets of matrix aerosol onto the sample surface for improved matrix homogeneity, reduced crystal size, and controlled solvent effects. This system was then applied to the analysis of histological slices of brains from mice with homozygous disruption of the hexb gene (hexb-/-), a model of Tay-Sachs and Sandhoff disease, versus the functionally normal heterozygote (hexb+/-) by imaging MALDI-MS. This allowed profiling and localization of many different lipid species, and of particular interest, ganglioside GM2, asialo-GM2 (GA2), and sulfatides (ST). The presence of these compounds was confirmed by analysis of brain extracts using electrospray ionization in conjunction with tandem mass spectrometry (MS/MS). The major fatty acid of the ceramide backbone of both GM2 and GA2 was identified as stearic acid (18:0) versus nervonic acid (24:1) for ST by both tissue-imaging MS and ESI-MS/MS. GM2 and GA2 were highly elevated in hexb-/- and were both localized in the granular cell region of the cerebellum. ST, however, was localized mainly in myelinated fiber (white matter) region of the cerebellum as well as in the brain stem with a relatively uniform distribution and had similar relative signal intensity for both hexb+/- and hexb-/- brain. It was also observed that there were distinct localizations for numerous other lipid subclasses; hence, imaging MALDI-MS could be used for "lipidomic" studies. These results illustrate the usefulness of tissue-imaging MALDI-MS with matrix deposition by OCN for histologic comparison of lipids in tissues such as brains from this mouse model of Tay-Sachs and Sandhoff disease.     

Determination of N-glycosylation sites and site heterogeneity in glycoproteins

An approach for the characterization of glycosylation sites and oligosaccharide heterogeneity in glycoproteins based on a combination of nonspecific proteolysis, deglycosylation, and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FT MS) is described. Glycoproteins were digested with Pronase yielding primarily glycopeptides and amino acids. Nonglycosylated peptide fragments were susceptible to complete Pronase digestion to their constituent amino acids. Steric hindrance prohibited the digestion of the peptide moiety attached to the glycan. Glycopeptides were desalted and concentrated using solid-phase extraction and analyzed by MALDI MS. The oligosaccharides were also analyzed by MALDI MS after releasing the glycans from glycoproteins using PNGase F. The peptide moiety of the glycopeptides was identified by subtracting the masses of the glycans derived from PNGase F treatment from the masses of the glycopeptides. The experimental strategy was validated using glycoproteins with known oligosaccharide structures, ribonuclease B and chicken ovalbumin. This procedure was then used to determine the N-glycosylation sites and site heterogeneity of a glycoprotein whose glycosylation pattern was unknown, namely, the Xenopus laevis egg cortical granule lectin. This procedure is useful for determining protein site heterogeneity and structural heterogeneities of the oligosaccharide moiety of glycoproteins.