High-throughput global peptide proteomic analysis by combining stable isotope amino acid labeling and data-dependent multiplexed-MS/MS

In this work, we describe the application of a stable isotope amino acid (lysine) labeling in conjunction with data-dependent multiplexed tandem mass spectrometry (MS/MS) to facilitate the characterization and identification of peptides from proteomic (global protein) digests. Lysine auxotrophic yeast was grown in the presence of 13C-labeled or unlabeled lysine and combined after harvesting in equal proportions. Endoproteinase LysC digestion of the cytosolic fraction produced a global proteomic sample, consisting of heavy/light labeled peptide pairs. Then data-dependent multiplexed-MS/MS was applied to simultaneously select and dissociate only labeled peptide ion pairs. The approach allows differentiation between N-terminal (e.g., b-type ions) and C-terminal fragment ions (e.g., y-type ions) in resulting tandem mass spectra, as well as the capability of differentiation between near-isobaric glutamine and lysine residues. We also describe the utility of peptide composition and fragment information to support peptide identifications and examine the potential application of lysine labeling for differential quantitative protein analysis.     

Characterization of variable regions of monoclonal antibodies by top-down mass spectrometry

A technique for rapid characterization of variable regions of monoclonal antibodies (mAb) is described. Several intact mAbs were analyzed on a Thermo-Fisher LTQ-Orbitrap high-resolution mass spectrometer (MS) by in-source fragmentation. In-source fragmentation has the unique advantage of fragmenting all charge states of a protein at the same time and, thus, greatly improves the sensitivity of the fragment ions over a true MS/MS experiment, where a single charge state is isolated and fragmented. In addition, immediate fragmentation of the protein before tertiary structure formation may also facilitate protein fragmentation. This technique has been proved very useful for top-down analysis of large proteins. In-source fragmentation of mAbs generated a series of fragment ions. In addition to some small b and y ions from the light chain and heavy chain in the low m/z region, a series of b ions corresponding to N-terminal 106-120 residues of both heavy chain and light chain were observed. The cleavage sites for these b ions happen to be near the linker regions between the variable domains and the constant domains of these antibodies. These b ions, therefore, correspond to the entire variable region of each chain. Similar results were obtained for all mAbs analyzed, including both immunoglobulin G1 and G2 molecules. To further characterize the variable regions, these b ions were isolated and fragmented by collision-induced dissociation in the linear trap, followed by mass analysis in the orbitrap. Large number of product ions was observed from these b ions. Many of these product ions are internal fragments between the two disulfide-linked cysteine residues. To demonstrate the capability of the technique, several mAbs were force-oxidized by treating with tert-butyl hydroperoxide, followed by mass spectrometric analysis. In-source fragmentation and MS/MS of the variable region b ions clearly identified the locations of the oxidized methionine.     

Approach for determining protein ubiquitination sites by MALDI-TOF mass spectrometry

Protein ubiquitination plays an important role in the degradation and other functional regulation of cellular proteins in organisms ranging from yeasts to mammals. Trypsin digestion of ubiquitin conjugated proteins produces diglycine branched peptides in which the C-terminal Gly-Gly fragment of ubiquitin is attached to the epsilon-amino group of a modified lysine residue within the peptide. This provides a platform for mapping ubiquitination sites using mass spectrometry. Here we report the development of a novel strategy for determining posttraslational protein ubiquitination based on the N-terminal sulfonation of diglycine branched peptides. In contrast to conventional tandem MS spectra of native tryptic peptides, MALDI MS/MS analysis of a sulfonated tryptic peptide containing a diglycine branch generates a unique spectrum composed of a signature portion and a sequence portion. The signature portion of the spectrum consists of several intense ions resulting from the elimination of the tags, the N-terminal residues at the peptide and the branch, and their combination. This unique ion distribution pattern can distinguish ubiquitination modificatons from others and can identify the first N-terminal residues of the peptides as well. The sequence portion consists of an exclusive series of y-type ions and y' ions (differing by the loss of one glycine residue from the sulfonated diglycine branch) that can directly reveal the amino acid sequence of the peptide and the precise location of the ubiquitination site. The technique is demonstrated for a series of synthetic peptides and is validated by a model protein, tetraubiquitin. Our results show that the MALDI MS/MS analysis of sulfonated tryptic peptides can provide a highly effective method for the determination of ubiquitination substrates, ubiquitination sites on protein targets, and modification sites on ubiquitins themselves.     

De novo sequencing of neuropeptides using reductive isotopic methylation and investigation of ESI QTOF MS/MS fragmentation pattern of neuropeptides with N-terminal dimethylation

A stable-isotope dimethyl labeling strategy was previously shown to be a useful tool for quantitative proteomics. More recently, N-terminal dimethyl labeling was also reported for peptide sequencing in combination with database searching. Here, we extend these previous studies by incorporating N-terminal isotopic dimethylation for de novo sequencing of neuropeptides directly from tissue extract without any genomic information. We demonstrated several new sequencing applications of this method in addition to the identification of the N-terminal residue using the enhanced a(1) ion. The isotopic labeling also provides easier and more confident de novo sequencing of peptides by comparing similar MS/MS fragmentation patterns of the isotopically labeled peptide pairs. The current study on neuropeptides shows several distinct fragmentation patterns after N-terminal dimethylation which have not been reported previously. The y((n-1)) ion is enhanced in multiply charged peptides and is weak or missing in singly charged peptides. The MS/MS spectra of singly charged peptides are simplified due to the enhanced N-terminal fragments and suppressed internal fragments. The neutral loss of dimethylamine is also observed. The mechanisms for the above fragmentations are proposed. Finally, the structures of the immonium ion and related ions of N(alpha), N(epsilon)-tetramethylated lysine and N(epsilon)-dimethylated lysine are explored.     

Dimethyl isotope-coded affinity selection for the analysis of free and blocked N-termini of proteins using LC-MS/MS

Because of enhanced a(1) ion signals, dimethyl labeling is useful for identifying the N-termini of proteins or peptides. Herein, we describe a novel strategy that uses dimethyl isotope-coded affinity selection (DICAS) to isolate peptides that contain either the dimethylated or in vivo blocked N-termini of proteins for comprehensive sequence analyses by LC-MS/MS. In this method, dimethyl labeling at the protein level was first performed using formaldehyde-d(2) to label all unblocked protein N-termini and lysine residues, followed by trypsin digestion. The free N-terminal amines of internal peptides generated by digestion were captured by solid supports with aldehyde functionalities through reductive amination. The flow-through fractions, which contained either in vivo or in vitro blocked N-terminal peptides, were subjected to sequence analyses by LC-MS/MS. Owing to the unique feature of a1 signal enhancement associated with dimethylated peptides and the use of the deuterium reagent, the in vitro blocked (or in vivo free) N-termini of proteins could be easily differentiated from the in vivo blocked N-termini. Thus, their sequences and N-terminal modifications could be assigned unambiguously from MS/MS spectra. In this study, the completeness of the labeling and the efficiency of the isolation method were first confirmed by the use of a mixture of model proteins composed of hemoglobin, myoglobin, and alpha-lactalbumin. The N-termini of all three proteins, including both alpha and beta chains of hemoglobin as well as a signal sequence located in the N-termini of alpha-lactalbumin, were successfully identified. The protocol was then applied to the analysis of an unfractionated lysate of MCF-7 cells. Results indicate that more than 80% of the isolated peptides contained the N-termini of unique proteins, and many of them were consistent with known or inferred N-terminal processing such as methionine removal and acetylation. In addition, using the DICAS approach, we identified a novel N-terminal formylation for the Ig kappa chain V-III region SIE protein and a novel N-terminal signal sequence (1th-32th amino acid) for profilin.     

Electrospray tandem mass spectrometry of intact beta-chain hemoglobin variants

In this report, we present data to illustrate how human hemoglobin (Hb) variants can be identified by electrospray tandem mass spectrometry (MS/MS) of the intact Hb chains following the one-step dilution of whole blood. MS/MS spectra were recorded on a series of intact beta-chain human Hb variants. The resultant spectra were interpreted, and using the information gleaned from the fragmentation patterns of known variants, two unknown beta-chain variants were characterized solely by this mass spectrometric method. Fragment ions that serve to identify beta-chain variants were identified. The fragmentation patterns of the intact beta-chain [M + 18H]18+ ions showed classical facile cleavages adjacent to acidic residues and N-terminal to proline residues, with Thr50-Pro51 being the most prominent cleavage site. Abundant product ions were formed by peptide bond cleavage in the regions close to the termini of the beta chain, the central region being less well-represented in the MS/MS spectra. Nearly 50% of the beta-chain primary structure could be determined by MS/MS of the intact chain. However, analysis of the Hb variants where mutations have occurred in the inner region (residues 58-111) of the beta globin proved to be difficult and required mass spectrometric analysis of their tryptic peptides for a complete identification.     

Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics

In glycoproteomics, key structural issues, protein identification, locations of glycosylation sites, and evaluation of the glycosylation site microheterogeneity should be easily evaluated in a large number of glycoproteins, while mass spectrometry (MS) provides substantial information about individual purified glycoproteins. Considering that structural issues are elucidated by studying glycopeptides and that the tandem MS of a tryptic peptide composed of several amino acid residues is enough for protein identification, construction of an MS-based method handling tryptic glycopeptides would be of considerable benefit in research. To this end, a simple and efficient method, utilizing hydrophilic binding of carbohydrate matrixes such as cellulose and Sepharose to oligosaccharides, was successfully applied to the isolation of tryptic glycopeptides. Both peptide and oligosaccharide structures were elucidated by multiple-stage tandem MS (MS(n)) of the ions generated by matrix-assisted laser desorption/ionization (MALDI), as follows. The MALDI ion trap mass spectrum of a tryptic glycopeptide mixture from N-linked glycoproteins was composed of the [M + H]+ ions of component glycopeptides. Collision-induced dissociation (CID) of the glycopeptide [M + H]+ ion generated saccharide-spaced peaks, with an interval of, for example, 146, 162, and 203 Da, and their fragment ions corresponding to the peptide and peptide + N-acetylglucosamine (GlcNAc) species in the MS2 spectrum. The saccharide-spaced ladder served to outline oligosaccharide structures, which were then selected as precursors for subsequent MS(n) analyses. The peptide or peptide + GlcNAc ions in the MS2 spectrum or the corresponding ions abundant in the MS1 spectrum were subjected to CID for determination of peptide sequences, to identify proteins and their glycosylation sites. The strategy, isolation of glycopeptides followed by MS(n) analysis, efficiently characterized the structures of beta2-glycoprotein I with four N-glycosylation sites and was applied to an analysis of total serum glycoproteins.     

Use of deuterium-labeled lysine for efficient protein identification and peptide de novo sequencing

Here, we describe a method for protein identification and de novo peptide sequencing. Through in vivo cell culturing, the deuterium-labeled lysine residue (Lys-d4) introduces a 4-Da mass tag at the carboxyl terminus of proteolytic peptides when cleaved by certain proteases. The 4-Da mass difference between the unlabeled and the deuterated lysine assigns a mass signature to all lysine-containing peptides in any pool of proteolytic peptides for protein identification directly through peptide mass mapping. Furthermore, it was used to distinguish between N- and C-terminal fragments for accurate assignments of daughter ions in tandem MS/MS spectra for sequence assignment. This technique simplifies the labeling scheme and the interpretation of the MS/MS spectra by assigning different series of fragment ions correctly and easily and is very useful in de novo peptide sequencing. We have also successfully implemented this approach to the analysis of protein mixtures derived from the human proteome.     

Assignment and quantification of 2-aminopyridine derivatized oligosaccharide isomers coeluted on reversed-phase HPLC/MS by MSn spectral library

2-Aminopyridine (PA)-derivatized oligosaccharides from IgG were analyzed by using reversed-phase HPLC/mass spectrometry (RP-HPLC/MS) and a MS(n) spectral library, in particular, focusing on two pairs of isomers incompletely separated or coeluted in chromatograms. We previously reported that MS(n) spectral matching considering both major fragment ions (m/z) and intensities is useful and applicable to the structural assignment of PA-oligosaccharide isomers. In this study, MS(n) spectral matching based on the MS(n) spectral library was applied to the assignment of these PA-oligosaccharide isomers in IgG. Its usefulness was investigated by comparing it to the conventional two-dimensional mapping method based on retention time indexes. Specifically, we focus on the assignment and quantification of the isomers, which are coeluted in chromatograms. From this, we propose a new method using MS(n) spectral matching and the working curve on which are plotted the relative intensities of selected fragment ions in their MS(2) spectra versus various mixtures of the isomers. This new method demonstrated that the obtained quantities coincide very well with those estimated after separating by a combination of lectin and reversed-phase columns. This means that separation by RP-HPLC/MS is greatly simplified because complete separation of the isomers is no longer required. Application of this new method was tested by using the two other pairs of fucosylated and nonfucosylated PA-oligosaccharides from IgG. The results showed that this method works for them as well.     

Complete mass spectral characterization of a synthetic ultralow-molecular-weight heparin using collision-induced dissociation

Glycosaminoglycans (GAGs) are a class of biologically important molecules, and their structural analysis is the target of considerable research effort. Advances in tandem mass spectrometry (MS/MS) have recently enabled the structural characterization of several classes of GAGs; however, the highly sulfated GAGs, such as heparins, have remained a relatively intractable class due their tendency to lose SO(3) during MS/MS, producing few sequence-informative fragment ions. The present work demonstrates for the first time the complete structural characterization of the highly sulfated heparin-based drug Arixtra. This was achieved by Na(+)/H(+) exchange to create a more ionized species that was stable against SO(3) loss, and that produced complete sets of both glycosidic and cross-ring fragment ions. MS/MS enables the complete structural determination of Arixtra, including the stereochemistry of its uronic acid residues, and suggests an approach for solving the structure of more complex, highly sulfated heparin-based drugs.     

193 nm ultraviolet photodissociation of imidazolinylated Lys-N peptides for de novo sequencing

The goal of many MS/MS de novo sequencing strategies is to generate a single product ion series that can be used to determine the precursor ion sequence. Most methods fall short of achieving such simplified spectra, and the presence of additional ion series impede peptide identification. The present study aims to solve the problem of confounding ion series by enhancing the formation of "golden" sets of a, b, and c ions for sequencing. Taking advantage of the characteristic mass differences between the golden ions allows N-terminal fragments to be readily identified while other ion series are excluded. By combining the use of Lys-N, an alternate protease, to produce peptides with lysine residues at each N-terminus with subsequent imidazolinylation of the ε-amino group of each lysine, peptides with highly basic sites localized at each N-terminus are generated. Subsequent MS/MS analysis by using 193 nm ultraviolet photodissociation (UVPD) results in enhanced formation of the diagnostic golden pairs and golden triplets that are ideal for de novo sequencing.     

Divinyl sulfone as a postdigestion modifier for enhancing the a(1) Ion in MS/MS and postsource decay: potential applications in proteomics

Divinyl sulfone reacts at pH 8-9 with the alpha-amino groups of N-terminal residues, proline, the epsilon-amino groups of lysine, and the histidine side chains of peptides. This reaction leads to great enhancement of the abundance of the normally weak or missing "a(1)" fragment ion in MS/MS analysis defining the N-terminal residue of a peptide in a digest. This provides "one-step Edman-like" information that, together with a fairly accurately determined mass, often enables one to correctly identify a protein or family of proteins. The applicability of this procedure in proteomics was demonstrated with several peptides and tryptic digests of protein mixtures by LC-MS/MS experiments using a QTOF and MALDI-PSD analyses. Advantages of this approach are its simple chemistry, retention of charge multiplicity, and possibly, shortening of database search time. Used with other MS/MS data, it provides higher confidence in the scores and identification of a protein found in peptide mass fingerprinting. Moreover, this approach has an advantage in "de novo" sequencing due to its ability to decipher the first amino acid of a peptide whose information is normally unavailable in MS/MS spectra.     

Study of the fragmentation mechanism of protonated 6-hydroxychlorzoxazone: application in simultaneous analysis of CYP2E1 activity with major human cytochrome P450s

The application of liquid chromatography tandem mass spectrometry for simultaneous analysis of major human cytochrome P450 activities via a single atmospheric pressure ionization (API) LC/MS/MS method has been hampered by the preferred detection of 6-hydroxychlorzoxazone (HCZ), the metabolite of the CYP2E1 probe, chlorzoxazone, under negative API. An initial simulation of the dissociation constants suggested the potential ionization of the enol form of HCZ at low pH, and the accurate mass measurements confirmed the presence of the protonated HCZ signal under (+) ESI at pH 3. However, the CID spectrum of the protonated HCZ resulted in a few intense, but uncommon, fragment ions that could be utilized for specific selected reaction monitoring (SRM) transitions. The deduced elemental compositions of these fragment ions indicated possible aromatic ring opening for the first two intense product ions at m/z 130 and 115, as well as chlorine radical loss for the third ion at m/z 151. Further precursor and product ion scan studies, along with the deuterium ion exchange in solution, revealed the involvement of three distinct pathways of fragmentation. The m/z 186-->130 transition, which was shown to be specific in human plasma and rat hepatic microsomes, was further combined with the SRM transition of reserpine (internal standard) and eight probe substrates for human cytochrome P450 isoforms. This led to the development of a full LC/MS/MS method capable of analyzing a total of nine human P450 activities within 3 min, including CYP2E1, using a single assay in the (+) ESI mode. The HCZ assay showed excellent linearity with a coefficient of determination (R2) greater than 0.98 at dynamic range of 0.05 (LOQ) to 40 microM. Preliminary data from the three-day validation of the HCZ assay indicated that the accuracy and precision for quality control samples was within +/- 15% of the spiked concentration at all levels.     

Ion activation in electron capture dissociation to distinguish between N-terminal and C-terminal product ions

We present a method to distinguish N-terminal from C-terminal product ions in electron capture dissociation (ECD) MS/MS due to the change in relative abundances of even-electron (prime) and odd-electron (radical) product ions produced in consecutive ECD and activated ion-ECD mass spectra. The method is based on the rate and direction of hydrogen atom transfer between N-terminal and C-terminal ECD products and its dependence on ion internal energy. We demonstrate that increasing ion internal energy by vibrational activation prior to ECD results in decreased ratio of radical/prime N-terminal product ions (c*/c' ratio), but increased ratio of radical/prime C-terminal product ions (z*/z' ratio) in many cases. The combination of AI-ECD and ECD promises to increase the confidence of mass spectrometry-based peptide sequencing and protein identification.     

T3-sequencing: targeted characterization of the N- and C-termini of undigested proteins by mass spectrometry

A novel extension of the "top-down" approach is introduced for the selective characterization of protein termini that does not involve proteolytic digestion steps. N- and C-terminal peptides were generated from intact proteins in the mass spectrometer and further analyzed by MS/MS-an approach referred to as T(3)-sequencing. N-terminal and C-terminal fragment ion series were obtained by the pseudo-MS/MS technique in-source decay (ISD) on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). These ions provided near-terminal sequence tags from the undigested protein in the ISD spectrum acquired in reflector mode and allowed to screen for the proper processing state of the terminus with respect to a reference sequence. In the second step of T(3)-sequencing, the precursor ions, which have been generated by ISD and which included the N- or C-terminal sequence, were selected in the timed ion gate of a MALDI-TOF/TOF mass spectrometer for MS/MS analysis. These spectra allowed identification of the protein, the proper definition of both termini, and allowed confirmation of suspected terminal modifications. T(3)-Sequencing appears to be an alternative to classical Edman sequencing, which is fast and even permits the analysis of N-terminally blocked proteins and their C-terminus.     

Automated monitoring of phosphatidylcholine biosyntheses in Plasmodium falciparum by electrospray ionization mass spectrometry through stable isotope labeling experiments

The metabolic pathways contributing to phosphatidylcholine biosyntheses in Plasmodium falciparum, the malaria-causing parasite, was explored by electrospray ionization mass spectrometry. Phosphatidylcholine produced by the CDP-choline pathway and by the methylation of phosphatidylethanolamine was identified and quantified through isotopic labeling experiments. A straightforward method based on cone voltage directed in-source fragmentations and relative abundance measurement of endogenous versus deuterated specific fragment ions was developed for simple and rapid automated data acquisition. Such high-throughput analytical protocol allowed us to measure the relative contribution of two different metabolic pathways leading to phosphatidylcholine without performing technically more demanding and time-consuming MS/MS or LC/MS experiments.     

Fluorescein as a versatile tag for enhanced selectivity in analyzing cysteine-containing proteins/peptides using mass spectrometry

Fluorescence-based tagging in proteomics is useful in tracking and quantifying target proteins during sample preparation or chromatographic processes. In this study, we report a novel cysteinyl tagging method using a popular fluorophore, fluorescein derivative. Such visible dyes were shown to have multiple unique characteristics, including a unique reporter ion containing the dye moiety caused by collision-induced dissociation (CID) and high affinity toward multicarboxylate functional groups, which could be useful for enhanced selectivity in MS-based proteomics. We used sulfhydryl-reactive 5-iodoacetamidofluorescein to target cysteinyl residues on the intact protein of ovalbumin and bovine serum albumin as well as proteins in MCF-7 cells. After trypsin digestion, the digests were analyzed by nanoLC-ESI-Q-TOF or MALDI-TOF. The resulting MS spectra of tryptic fragments were similar to those of unlabeled or iodoacetamide-derivatized proteins, and the MS/MS fragmentation of all fluorescein-tagged peptides was readily interpretable with intact label. Thus, fluorescein-derivatized proteins can be identified by automatic mass mapping or peptide sequencing with high confidence. It is notable that, in MS/MS mode, a strong reporter ion (m/z 422) containing the fluorescein moiety was readily detected and was believed to derive from the immonium fragment of fluorescein-labeled cysteine residues, f C (m/z 463), under CID conditions. Using a precursor scan of the reporter ion, a cysteinyl protein, ovomucoid, was identified to be present in the ovalbumin sample as an impurity. The fluorescein derivatives were further shown to have high affinities toward metal-chelating materials that have iminodiacetic acid functional groups either with or without the presence of bound metal ions. When coupling with stable isotope dimethyl labeling, fluorescein-tagged peptides could be selectively enriched, identified, and quantified. In view of its popularity, visible tracking, and unique characteristics for developing selective methods, fluorescein tagging holds great promises for targeting proteomics.     

Identification of protein ubiquitylation by electrospray ionization tandem mass spectrometric analysis of sulfonated tryptic peptides

We report here the application of electrospray ionization tandem mass spectrometry for the characterization of protein ubiquitylation, an important posttranslational modification of cellular proteins. Trypsin digestion of ubiquitin-conjugated proteins produces diglycine branched peptides containing the modification sites. Chemical derivatization by N-terminal sulfonation was carried out on several model peptides for the formation of a characteristic fragmentation pattern in their MS/MS analysis. The fragmentation of derivatized singly charged peptides results in a product ion distribution similar to that already observed by MALDI-TOF MS/MS. Signature fragments distinguished the diglycine branched peptides from other modified and unmodified peptides, while the sequencing product ions reveal the amino acid sequence and the location of the ubiquitylation site. Doubly charged peptide derivatives fragment in a somewhat different manner, but several fragments characteristic to diglycine branched peptides were observed under low collision energy conditions. These signature peaks can also be used to identify peptides containing ubiquitylation sites. In addition, a marker ion corresponding to a glycine-modified lysine residue produced by high-energy fragmentation provides useful information for identity verification. The method is demonstrated by the analysis of three ubiquitin-conjugated proteins using LC/MS/MS.     

Observation of hydrogen-deuterium exchange of ubiquitin by direct analysis of electrospray capillary-skimmer dissociation with Fourier transform ion cyclotron resonance mass spectrometry

The structure of ubiquitin, a small cytoplasmic protein with an extended beta-sheet and an alpha-helix surrounding a hydrophobic core, has been characterized by hydrogen-deuterium (H/D) exchange labeling in conjunction with successive analysis by capillary-skimmer dissociation with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium content of each fragment ion was investigated at different times, and the results indicate that the deuterium incorporation rate into the backbone amides of ubiquitin varied depending on the environment of the amide hydrogens. Amide hydrogens of the N-terminal beta-strand showed quite slow exchange while those of the 35-39 loop were exchanged within a short exposure time to deuterium oxide. It was also possible to evaluate the difference in hydrogen-bond stability. The present data are consistent with the structural features obtained by X-Ray and NMR analyses. Although some of the labeling information might be lost by the scrambling of amide protons during capillary-skimmer dissociation, the results demonstrate that the present method provides useful higher-order structural information for proteins.