Identification of GlcNAcylation sites of peptides and alpha-crystallin using Q-TOF mass spectrometry

The addition of a single N-acetylglucosamine residue O-linked to serine and threonine residues of nuclear and cytoplasmic proteins is a widespread modification throughout all eukaryotes. The conventional method for detecting and locating sites of modification is a multi-step radioactivity-based protocol. In this paper we show that using quadrupole time-of-flight (Q-TOF) mass spectrometry, modification sites can be identified at a significantly higher sensitivity than previous approaches. This is the first demonstration that sites of O-GlcNAcylation can be identified directly using mass spectrometry.     

Matrix-assisted laser desorption/ionization directed nano-electrospray ionization tandem mass spectrometric analysis for protein identification

In those cases where the information obtained by peptide mass fingerprinting or matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) is not sufficient for unambiguous protein identification, nano-electrospray ionization (nano-ESI) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis must be performed. The sensitivity of nano-ESI/MS, however, is lower than that of MALDI-MS, especially at very low analyte concentrations and/or in the presence of contaminants, such as salt and detergents. Moreover, to perform ESI-MS/MS, the peptide masses of the precursor ions must be known. The approach described in this paper, MALDI-directed nano-ESI-MS/MS, makes use of information obtained from the more sensitive MALDI-MS experiments in order to direct subsequent nano-ESI-MS/MS experiments. Peptide molecular ions found in the MALDI-MS analysis are then selected, as their (+2) precursor ions, for nano-ESI-MS/MS sequencing, even though these ions cannot be detected in the ESI-MS spectra. This method, originally proposed by Tempst et al. (Anal. Chem. 2000, 72: 777-790), has been extended to provide better sensitivity and shorter analysis times; also, a comparison with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been performed. These experiments, performed using quadrupole time-of-flight instruments equipped with commercially available nano-ESI sources, have allowed the unambiguous identification of in-gel digested proteins at levels below their ESI-MS detection limits, even in the presence of salts and detergents.     

Analysis of post-translational modification and characterization of the domain structure of dynamin A from Dictyostelium discoideum

The post-translational modifications of the 96 kDa protein dynamin A from Dictyostelium discoideum were analyzed using Q-TOF mass spectrometry. The accurate molecular mass of the intact protein revealed a covalent modification causing an additional mass of 42 Da. The modification could be identified as N-terminal acetylation by tandem mass spectrometry. Extracted ion chromatograms for the a(1) and b(1) ion of the tryptic T1 peptide were used to detect the acetylated peptide within 54 nanoelectrospray ionization tandem mass spectra. Owing to the accurate molecular mass of the intact protein, additional covalent modifications could be excluded. In addition to the covalent modification, the domain structure of dynamin A was determined by applying a combination of limited proteolysis, sodium dodecylsulfate polyacrylamide gel electrophoresis, automated tandem mass spectrometry and protein database searching.     

Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation

Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.     

Live single-cell molecular analysis by video-mass spectrometry.

The direct molecular analysis of a live single cell viewed under a video-microscope has been developed. The cell contents are sucked into a nano-electrospray tip, and hundreds of peaks of ionic compounds of low molecular weight are detected by nano-ESI Q-TOF mass spectrometry (MS). Cell-specific MS peaks in a single mouse-embryonic fibroblasts cell are extracted by a t-test, and one of the peaks is proceeded to MS/MS analysis for molecular identification. This method is direct and quick to identify the molecules of a cell with simultaneous observation by a video-microscope.     

Mapping low-resolution three-dimensional protein structures using chemical cross-linking and Fourier transform ion-cyclotron resonance mass spectrometry

Techniques in mass spectrometry (MS) combined with chemical cross-linking have proven to be efficient tools for the rapid determination of low-resolution three-dimensional (3-D) structures of proteins. The general procedure involves chemical cross-linking of a protein followed by enzymatic digestion and MS analysis of the resulting peptide mixture. These experiments are generally fast and do not require large quantities of protein. However, the large number of peptide species created from the digestion of cross-linked proteins makes it difficult to identify relevant intermolecular cross-linked peptides from MS data. We present a method for mapping low-resolution 3-D protein structures by combining chemical cross-linking with high-resolution FTICR (Fourier transform ion-cyclotron resonance) mass spectrometry using cytochrome c and hen egg lysozyme as model proteins. We applied several homo-bifunctional, amine-reactive cross-linking reagents that bridge distances from 6 to 16 A. The non-digested cross-linking reaction mixtures were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to determine the extent of cross-linking. Enzymatically digested reaction mixtures were separated by nano-high-performance liquid chromatography (nano-HPLC) on reverse-phase columns applying water/acetonitrile gradients with flow rates of 200 nL/min. The nano-HPLC system was directly coupled to an FTICR mass spectrometer equipped with a nano-ESI (electrospray ionization) source. Cross-linking products were identified using a combination of the GPMAW software and ExPASy Proteomics tools. For correct assignment of the cross-linking products the key factor is to rely on a mass spectrometric method providing both high resolution and high mass accuracy, such as FTICRMS. By combining chemical cross-linking with FTICRMS we were able to rapidly define several intramolecular constraints for cytochrome c and lysozyme.     

Microfluidic electrocapture interfaced with electrospray mass spectrometry

Microfluidic electrocapture of peptides and proteins in an inert capillary with electric contacts via conductive membranes is useful for sample handling before mass spectrometry. The use of electrocapture has already been demonstrated for sample clean-up, pre-concentration, chemical modification and peptide separation, all without the need for supporting gels or chemical binding. This method allows multiple micro-reactions, extensive peptide separations and work with membrane proteins from detergent-solubilized samples. Until now, electrocapture has utilized MALDI mass spectrometry, but here we demonstrate that it can be interfaced with electrospray ionization and hence with on-line mass spectrometric analysis of peptides separated from protein digests. These applications combined with the present on-line approach show electrocapture to be a versatile technology with great potential.     

Mass spectrometric approaches for the characterization of proteins on a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer

This study demonstrates structural and conformational characterization of proteins by nanoflow electrospray ionization (nanoESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) utilizing a quadrupole time-of-flight (Q-TOF) mass spectrometer (Micromass, Manchester, England). Model peptides were successfully sequenced at the 35 attomole (amol) level, and peptides derived from a tryptic in-gel digest of 25 femtomole (fmol) bovine serum albumin (BSA) were successfully sequenced. The results demonstrated that the MS/MS sensitivity of the Q-TOF clearly surpassed the detection limit of the silver stain. A silver destaining step greatly improved the mass analysis of peptides derived from in-gel digests. Interestingly, sequence analysis revealed BSA residue 424 (tyrosine) as a potential chlorination site. In addition, a modified procedure was successfully used to extract and measure the masses of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-resolved proteins in the 10-68.5 kDa range. The Q-TOF was also used to monitor conformational changes of proteins. These experiments demonstrated an acid-induced denaturation of BSA in the pH 3-4 range, and heat-induced unfolding of cytochrome c between 50 and 60 degrees C. Finally, Zn2+ binding was demonstrated for the carbonic anhydrase apoprotein. In summary, the wide range of applications and the high quality of the experimental data made the Q-TOF mass spectrometer a powerful analytical tool for protein characterization.     

Accurate mass measurement in nano-electrospray ionization mass spectrometry by alternate switching of high voltage between sample and reference sprayers

An electrospray dual sprayer, which generates separate sample and reference sprays by alternately switching the high voltage between the two sprayers, is described. The technique permits accurate mass measurements in nano-electrospray ionization mass spectrometry (ESI-MS) to be obtained using a quadrupole/orthogonal acceleration time-of-flight mass spectrometer (Q-TOF). Similar to the method employed with a dual ESI source (Wolff JC et al., Anal. Chem. 2001; 73: 2605), the two sprays are orthogonal with respect to each other, but can be independently sampled without any baffle between these sprays. The reference sprayer is used in the original configuration of the ESI source and was optimized for a 1-2 muL/min flow, whereas the sample sprayer can be either a conventional glass capillary or a borosilicate tip of the type used for nano-ESI. Both sprayers can be positioned close to the cone so as to give maximum ion currents. The sample and reference sprays are independently generated by raising the potentials on the sample and reference sprayers to 1.4 and 3.0 kV, respectively; the high voltages can be rapidly turned on and off in ca. 1 ms. A nano-ESI-MS or nano-flow LC/ESI-MS experiment using a Q-TOF coupled with the above system gave mass accuracies within 3 ppm for measurements of ions up to m/z 1000 using subpicomole samples.     

Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin

Recombinant human erythropoietin (rhEPO) has been extensively used as a pharmaceutical product for treating anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics, and in-vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple-enzyme digestion, hydrophilic-interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high-resolution and high-accuracy (HR–AM) mass spectrometry using an Orbitrap. In total, 74 intact glycopeptides from four glycosylation sites at N₂₄, N₃₈, N₈₃, and O₁₂₆ were identified, with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR–AM data enabled relative quantification of glycoforms. Our results could be extended to quality control of rhEPO or could help establish detection approaches for glycosylation of other proteins. Graphical Abstract

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Detection and identification of arginine modifications on methylglyoxal-modified ribonuclease by mass spectrometric analysis

Analysis of the broad range of trace chemical modifications of proteins in biological samples is a significant challenge for modern mass spectrometry. Modification at lysine and arginine residues, in particular, causes resistance to digestion by trypsin, producing large tryptic peptides that are not readily sequenced by mass spectrometry. In this work, we describe the analysis of ribonuclease (RNase) modified by methylglyoxal (MGO) under physiological conditions. For detection of modifications, we use comparative analysis of the single combined spectra extracted from the full-scan MS data of the tryptic digests from native and modified proteins. This approach revealed 11 ions unique to MGO-modified RNase, including a 32-amino acid peptide containing a modified Arg-85 residue. Sequential digestion of MGO-modified RNase by endoproteinase Glu-C and trypsin was required to obtain peptides that were amenable to sequencing analysis. Arg-39 was identified as the main site of modification (35% modification) on MGO-modified Rnase, and the dihydroxyimidazolidine and hydroimidazolone derivatives were the main adducts formed, with minor amounts of the tetrahydropyrimidine and argpyrimidine derivatives. For identification of these products, we used variations in source voltage and collision energy to obtain the dehydration and decarboxylation products of the tetrahydropyrimidine-containing peptides and dehydration of the dihydroxyimidazoline-containing peptides. The resultant spectra were dependent on the cone voltage and collision energy, and analysis of spectra at various settings permitted structural assignments. These studies illustrate the usefulness of single combined mass spectra extracted from full-scan data and variations in source and collision cell voltages for detection and structural characterization of chemical adducts on proteins.     

Isotope-labeled cross-linkers and fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex

For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an "IQ-like motif." Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d0 and d4) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central alpha-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural data of protein assemblies within a few days.     

Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140DeltaCFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis.     

Structural analysis of a glycoprotein by liquid chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry. Application to recombinant human thrombomodulin

Using recombinant human thrombomodulin (rhTM) expressed in Chinese hamster ovary (CHO) cells, we studied the structural analysis of a glycoprotein by liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS-MS). First, we analyzed the structure of both the O- and N-linked glycans in rhTM by oligosaccharide mapping using LC-MS equipped with a graphitized carbon column (GCC-LC-MS). Major O- and N-linked glycans were determined to be core 1 structure and fucosyl biantennary containing NeuAc(0-2) respectively. Next, the post-translational modifications and their heterogeneities, including the site-specific glycosylation, were analyzed by mass spectrometric peptide/glycopeptide mapping of trypsin-digested rhTM and precursor-ion scanning. Precursor-ion scanning was successful in the detection of five glycopeptides. Four N-glycosylation sites and their site-specific carbohydrate heterogeneity were determined by their mass spectra. O-Glycosylation could be estimated on the basis of its mass spectrum. We were able to identify partial beta-hydroxylation on Asn324 and Asn439, and O-linked glucose on Ser287 from the peptide/glycopeptide map and their mass spectra. We demonstrated that a sequential analysis of LC-MS and LC-MS-MS are very useful for the structural analysis of O- and N-linked glycans, polypeptides, and post-translational modifications and their heterogeneities, including site-specific glycosylation in a glycoprotein. Our method can be applied to a glycoprotein in biological samples.     

Multidimensional nano-HPLC for analysis of protein complexes

The analysis of macromolecular protein complexes is an important factor in understanding most cellular processes, e.g., protein transport into cell organells, signal transduction via biological membranes, apoptosis, energy metabolism, directed motion of cells, and cell division. These complexes are not only built of various numbers of different proteins but also of prosthetic groups and RNA molecules. To understand the role each protein plays in a complex, a complete analysis of all protein compounds is necessary. Therefore, several separation steps have to be coupled to mass spectrometry to identify the proteins. In this work, we describe the application of multidimensional liquid chromatography, SCX-RP-LC as well as SAX-RP-LC, coupled to electrospray ion trap mass spectrometry. Tryptic digested ribosomes were separated by ion exchange chromatography manually collected and prepared for reversed phase chromatography to analyze the peptides via nano-ESI mass spectrometry. The total numbers of identified proteins are compared in consideration of the separation method (SCX-RP versus SAX-RP).     

StavroX—A Software for Analyzing Crosslinked Products in Protein Interaction Studies

Chemical crosslinking in combination with mass spectrometry has matured into an alternative approach to derive low-resolution structural information of proteins and protein complexes. Yet, one of the major drawbacks of this strategy remains the lack of software that is able to handle the large MS datasets that are created after chemical crosslinking and enzymatic digestion of the crosslinking reaction mixtures. Here, we describe a software, termed StavroX, which has been specifically designed for analyzing highly complex crosslinking datasets. The StavroX software was evaluated for three diverse biological systems: (1) the complex between calmodulin and a peptide derived from Munc13, (2) an N-terminal ß-laminin fragment, and (3) the complex between guanylyl cyclase activating protein-2 and a peptide derived from retinal guanylyl cyclase. We show that the StavroX software is advantageous for analyzing crosslinked products due to its easy-to-use graphical user interface and the highly automated analysis of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data resulting in short times for analysis. StavroX is expected to give a further push to the chemical crosslinking approach as a routine technique for protein interaction studies.     

PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry

A number of different approaches have been described to identify proteins from tandem mass spectrometry (MS/MS) data. The most common approaches rely on the available databases to match experimental MS/MS data. These methods suffer from several drawbacks and cannot be used for the identification of proteins from unknown genomes. In this communication, we describe a new de novo sequencing software package, PEAKS, to extract amino acid sequence information without the use of databases. PEAKS uses a new model and a new algorithm to efficiently compute the best peptide sequences whose fragment ions can best interpret the peaks in the MS/MS spectrum. The output of the software gives amino acid sequences with confidence scores for the entire sequences, as well as an additional novel positional scoring scheme for portions of the sequences. The performance of PEAKS is compared with Lutefisk, a well-known de novo sequencing software, using quadrupole-time-of-flight (Q-TOF) data obtained for several tryptic peptides from standard proteins.     

Mass spectrometric approach for the analysis of food proteins

In the study of food proteins, the need for accurate protein structural analysis has been acknowledged because of the fact that nucleotide sequencing alone is of limited analytical value if not combined with relevant information regarding the specific protein expressed and the occurrence of phosphorylation, glycosylation and disulphide bridges, and with the modification induced by the technological treatment. Mass spectrometry, whether used alone or to complement the traditional molecular-based techniques has become fundamental to the structural analysis of proteins. It is, moreover, virtually irreplaceable in determining post-translational modifications as conventional methods cannot deliver reliable data. What lies at the root of this methodological breakthrough is the combination of high-resolution separation techniques such as two-dimensional electrophoresis or capillary reverse- phase high-performance liquid chromatography with mass spectrometric analysis, what is termed "proteomic" analysis. Thus, it appears appropriate to state that the new mass spectrometric techniques have been established as a valuable and efficient tool for protein and peptide analysis in complex mixtures, like those from food matrices, enabling us therefore to provide accurate information on molecular weight and also to put forth a structural assessment at a low-picomole level of material. Thus, a series of alternative approaches have been developed based on advanced mass spectrometric analysis in conjunction with classic protein chemistry in order to provide an in-depth view of food protein structure. This review outlines several of these novel methodologies as they apply to structural characterization of food products.     

纳升电喷雾毛细管液相色谱-串联质谱鉴定K562细胞株中混合蛋白质

用标准蛋白质混合物建立了一种适用于低丰度混合蛋白质及其异构体分离与鉴定的蛋白质组学方法。通过IPG胶条等电聚焦分离蛋白质,染色后进行混合胶内酶切,采用纳升电喷雾毛细管液相色谱一串联质谱“散弹法（shot-gun）”分析酶切产物,并进行数据库检索鉴定蛋白质。运用该方法从K562细胞株样品中鉴定出14种具有重要功能的蛋白质,部分蛋白质同时在多个条带中出现,可能是异构体。肽段及其碎片离子的平均质量偏差小于0．05U,综合得分大都远远超过有效值。该方法灵敏、准确度高、分辨率高、省时、便于操椎存苍宗罾白甩异构体青而右优势．     

A comprehensive and non‐prefractionation on the protein level approach for the human urinary proteome: touching phosphorylation in urine

Increasing attention has been paid to the urinary proteome because it holds the promise of discovering various disease biomarkers. However, most of the urine proteomics studies routinely relied on protein pre‐fractionation and so far did not present characterization on phosphorylation status. Two robust approaches, integrated multidimensional liquid chromatography (IMDL) and Yin‐yang multidimensional liquid chromatography (MDLC) tandem mass spectrometry, were recently developed in our laboratory, with high‐coverage identification of peptide mixtures. In this study, we adopted a strategy without pre‐fractionation on the protein level for urinary proteome identification, using both the IMDL and the Yin‐yang MDLC methods for peptide fractionation followed by identification using a linear ion trap‐orbitrap (LTQ‐Orbitrap) mass spectrometer with high resolution and mass accuracy. A total of 1310 non‐redundant proteins were highly confidently identified from two experiments, significantly including 59 phosphorylation sites. More than half the annotated identifications were membrane‐related proteins. In addition, the lysosomal as well as kidney‐associated proteins were detected. Compared with the six largest datasets of urinary proteins published previously, we found our data included most of the reported proteins. Our study developed a robust approach for exploring the human urinary proteome, which would provide a catalogue of urine proteins on a global scale. It is the first report, to our best knowledge, to profile the urinary phosphoproteome. This work significantly extends current comprehension of urinary protein modification and its potential biological significance. Moreover, the strategy could further serve as a reference for biomarker discovery. Copyright © 2010 John Wiley & Sons, Ltd.