Coumarin tags for improved analysis of peptides by MALDI-TOF MS and MS/MS. 1. Enhancement in MALDI MS signal intensities

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF MS and MS/MS. The proposed tags, commercially available fluorescent derivatives of coumarin, can be advantageous for peptide analysis in both MS and MS/MS modes. This paper, part 1, will focus on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. Labeling peptides with tags containing the coumarin core was found to enhance the intensities of peptide peaks (in some cases over 40-fold) in MALDI-TOF MS using CHCA and 2,5-DHAP matrixes. The signal enhancement was found to be peptide- and matrix-dependent, being the most pronounced for hydrophilic peptides. No correlation was found between the UV absorptivity of the tags at the excitation wavelengths typical for UV-MALDI and the magnitude of the signal enhancement. Interestingly, peptides labeled with Alexa Fluor 350, a coumarin derivative containing a sulfo group (i.e., bearing strong negative charge), showed a 5-15-fold increase in intensity of MALDI MS signal in the positive ion mode, relative to the underivatized peptides, when 2,5-DHAP was used as the matrix. The Alexa Fluor 350 tag yielded a significantly higher signal relative to that for the CAF tag, likely due to the increased hydrophobicity of the coumarin structure. With 2,5-DHB, a decrease of MALDI MS signal was observed for all coumarin-labeled peptides, again relative to the unlabeled species. These findings support the hypothesis that derivatization with coumarin, a relatively hydrophobic structure, improves incorporation of hydrophilic peptides into hydrophobic MALDI matrixes, such as CHCA and 2,5-DHAP.     

Enrichment and identification of cysteine-containing peptides from tryptic digests of performic oxidized proteins by strong cation exchange LC and MALDI-TOF/TOF MS

The extreme complexity of sample and uninformative fragmentation of peptides in MS/MS experiments are two of several real challenges faced by proteomics. In this work, a strategy aimed at tackling these two problems is presented. Briefly, proteins were first oxidized by performic acid to cleave the disulfide bonds and simultaneously convert cysteine residue into its sulfonic form. Then the resultant sulfonic peptides were enriched by SCX chromatography, exploiting the negative solution charge of sulfonic group. The sulfonic peptide could be easily detected by MALDI-MS in negative mode and showed both enhanced fragmentation efficiency and a simplified spectrum in MALDI-MS/MS experiment in positive mode. The strength of the strategy was demonstrated by applying it to bovine serum albumin. Potential use of the strategy in proteomics was also discussed.     

Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS

A novel MS/MS-based analysis strategy using isotopomer labels, referred to as "tandem mass tags" (TMTs), for the accurate quantification of peptides and proteins is described. The new tags are designed to ensure that identical peptides labeled with different TMTs exactly comigrate in all separations. The tags require novel methods of quantification analysis using tandem mass spectrometry. The new tags and analysis methods allow peptides from different samples to be identified by their relative abundance with greater ease and accuracy than other methods. The new TMTs permit simultaneous determination of both the identity and relative abundances of peptide pairs using a collision induced dissociation (CID)-based analysis method. Relative abundance measurements made in the MS/MS mode using the new tags are accurate and sensitive. Compared to MS-mode measurements, a very high signal-to-noise ratio is achieved with MS/MS based detection. The new tags should be applicable to a wide variety of peptide isolation methods.     

Use of peptide retention time prediction for protein identification by off-line reversed-phase HPLC-MALDI MS/MS

A new algorithm, sequence-specific retention calculator, was developed to predict retention time of tryptic peptides during RP HPLC fractionation on C18, 300-A pore size columns. Correlations of up to approximately 0.98 R2 value were obtained for a test library of approximately 2000 peptides and approximately 0.95-0.97 for a variety of real samples. The algorithm was applied in conjunction with an exclusion protocol based on mass (15 ppm tolerance) and retention time (2-min tolerance for 0.66% acetonitrile/min gradient), MART criteria to significantly reduce the instrument time required for complete MS/MS analysis of a digest separated by RP HPLC. This was confirmed by reanalyzing the set of HPLC-MALDI MS/MS data with no loss in protein identifications, despite the number of virtually executed MS/MS analyses being decreased by 57%.     

IMS-IMS and IMS-IMS-IMS/MS for separating peptide and protein fragment ions

Multidimensional ion mobility spectrometry (IMS-IMS and IMS-IMS-IMS) techniques have been combined with mass spectrometry (MS) and investigated as a means of generating and separating peptide and protein fragment ions. When fragments are generated inside a drift tube and then dispersed by IMS prior to MS analysis, it is possible to observe many features that are not apparent from MS analysis alone. The approach is demonstrated by examining fragmentation patterns arising from electrospray ion distributions of insulin chain B and ubiquitin. The multidimensional IMS approach makes it possible to select individual components for collisional activation and to disperse fragments based on differences in mobility prior to MS analysis. Such an approach makes it possible to observe many features not apparent by MS analysis alone.     

Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards

Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standards. Synthetic peptides are designed to mimic peptides that are cleaved from target analyte proteins by proteolytic or chemical digestion, and the peptides selected serve as standards for quantification by LC/MS/MS on a triple quadrupole mass spectrometer. The technique is complementary to relative quantification techniques in widespread use by providing absolute quantitation of selected targets with greater sensitivity, dynamic range, and precision. Proteins that are found to be of interest by global proteome searches can be selected as targets for quantitation by the present method. This method has a much shorter analytical cycle time (minutes versus hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach using synthetic peptides as standards, in conjunction with proteolytic or chemical cleavage of target proteins, allows mass spectrometry to be used as a highly selective detector for providing absolute quantification of proteins for which no standards are available. We demonstrate that quantification is simple and reliable for the integral membrane protein rhodopsin with reasonable recoveries for replicate experiments using low-micromolar solutions of rhodopsin from rod outer segments.     

Artificial neural network analysis for evaluation of peptide MS/MS spectra in proteomics

The aim of the work was to explore usefulness of artificial neural network (ANN) analysis for the evaluation of proteomics data. The analysis was applied to the data generated by the widely used protein identification program Sequest, completed with several structural parameters readily calculated from peptide molecular formulas. Proteins from yeast cells were identified based on the MS/MS spectra of peptides. The constructed ANN was demonstrated to classify automatically as either "good" or "bad" the peptide MS/MS spectra otherwise classified manually. An appropriately trained ANN proves to be a high-throughput tool facilitating examination of Sequest's results. ANNs are recommended as a means of automatic processing of large amounts of MS/MS data, which normally must be considered in the analysis of complex mixtures of proteins in proteomics.     

A calibration method that simplifies and improves accurate determination of peptide molecular masses by MALDI-TOF MS

The use of delayed ion extraction in MALDI time-of-flight mass spectrometry distorts the linear relationship between m/z and the square of the ion flight time (t2) with the consequence that, if a mass accuracy of 10 ppm or better is to be obtained, the calibrant signals have to fall close to the analyte signals. If this is not possible, systematic errors arise. To eliminate these, a higher-order calibration function and thus several calibrant signals are required. For internal calibration, however, this approach is limited by signal suppression effects and the increasing chance of the calibrant signals overlapping with analyte signals. If instead the calibrants are prepared separately, this problem is replaced by an other; i.e., the ion flight times are dependent on the sample plate position. For this reason, even if the calibrants are placed close to the sample, the mass accuracy is not improved when a higher-order calibration function is applied. We have studied this phenomenon and found that the relative errors, which result when moving from one sample to the next, are directly proportional to m/z. Based on this observation, we developed a two-step calibration method, that overcomes said limitations. The first step is an external calibration with a high-order polynomial function used for the determination of the relation between m/z and t2, and the second step is a first-order internal correction for sample position-dependent errors. Applying this method, for instance, to a mass spectrum of a mixture of 18 peptides from a tryptic digest of a recombinant protein resulted in an average mass error of 1.0 ppm with a standard deviation of 3.5 ppm. When instead using a conventional two-point internal calibration, the average relative error was 2.2 ppm with a standard deviation of 15 ppm. The new method is described and its performance is demonstrated with examples relevant to proteome research.     

Continuous pH/salt gradient and peptide score for strong cation exchange chromatography in 2D-nano-LC/MS/MS peptide identification for proteomics

Tryptic digests of human serum albumin and human lung epithelial cell lysates were used as test samples in a novel proteomics study. Peptides were separated and analyzed using 2D-nano-LC/MS/MS with strong cation exchange (SCX) and reversed-phase chromatography and continuous gradient elution. The peptide elution conditions combined simultaneous pH gradient with ammonium acetate salt gradient elution modes. A novel empirical SCX peptide elution score was developed, which accounts for both the number of basic and acidic residues and, in part, their location within a sequence of a peptide. Average scores calculated for the fractionated peptide sequences correlated well with the pH of SCX elution fractions. Multiple peptides with identical amino acid sequences, but differing in cysteine tags possessing different positive charge and different SCX elution properties, were obtained by subjecting the samples to reduction and alkylation with different cysteine alkylating reagents: iodoacetamide, 4-vinylpyridine, and (3-acrylamidopropyl) trimethylammonium chloride. The structurally similar peptides were used as elution standards.     

HPLC and MALDI-TOF MS analysis of highly branched polystyrene: resolution enhancement by branching

Temperature gradient interaction chromatography (TGIC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were applied for the characterization of highly branched polystyrenes (PS) prepared by linking living polystyryl anions using 4-chlorodimethylsilylstyrene. Reversed-phase (RP)-TGIC showed an unexpectedly high resolution according to the number of branches despite significant overlap of the molecular weight as confirmed by MALDI-TOF MS. The enhancement of the resolution is ascribed to the contribution of the nonpolar groups in the branched PS: the dimethylsilyl groups in the branching unit as well as the sec-butyl initiator groups. As the number of branches increases, the number of nonpolar groups increases, which in turn increases the RP-TGIC retention synergistically with increasing molecular weight. In contrast, a poorer resolution was found in normal-phase-TGIC, in which the nonpolar groups reduce the retention. The resolution in RP-TGIC appears superior to that of liquid chromatography at the chromatographic critical condition (LCCC) of PS. It is seemingly due to the synergistic contribution of the incremental PS molecular weight to the functionality in the branched PS in RP-TGIC while only the functionality contributes to the separation in LCCC. This type of resolution enhancement could be utilized efficiently for the analysis of highly branched polymers such as dendrimers or hyperbranched polymers.     

On-plate selective enrichment and self-desalting of peptides/proteins for direct MALDI MS analysis

In this paper, a new technique has been proposed to achieve simultaneous peptides/proteins enrichment and wash-free self-desalting on a novel sample support with a circle hydrophobic-hydrophilic-hydrophobic pattern. Upon deposition, the sample solution is first concentrated in a small area by repulsion of the hydrophobic outer layer, and then, the peptides/proteins and coexisting salt contaminants are selectively captured in different regions of the pattern through strong hydrophobic and hydrophilic attractions, respectively. As a result, the detection sensitivity is improved by 2 orders of magnitude better than the use of the traditional MALDI plate, and high-quality mass spectra are obtained even in the presence of NaCl (1 M), NH(4)HCO(3) (100 mM), or urea (1 M). The practical application of this method is further demonstrated by the successful analysis of myoglobin digests with high sequence coverage, demonstrating the great potential in proteomic research.     

Identification and quantification of cardiac glycosides in blood and urine samples by HPLC/MS/MS.

Cardiac glycosides (CG) are of forensic importance because of their toxicity and the fact that very limited methods are available for identification of CG in biological samples. In this study, we have developed an identification and quantification method for digoxin, digitoxin, deslanoside, digoxigenin, and digitoxigenin by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS). CG formed abundant [M + NH4]+ ions and much less abundant [M + H]+ ions as observed with electrospray ionization (ESI) source and ammonium formate buffer. Under mild conditions for collision-induced dissociation (CID), each [M + NH4]+ ion fragmented to produce a dominant daughter ion, which was essential to the sensitive method of selected reaction monitoring (SRM) quantification of CG achieved in this study. SRM was compared with selected ion monitoring (SIM) regarding the effects of sample matrixes on the methodology. SRM produced lower detection limits with biological samples than SIM, while both methods produced equal detection limits with CG standards. On the basis of the HPLC/MS/MS results for CG, we have proposed some generalized points for conducting sensitive SRM measurements, in view of the property of analytes as well as instrumental conditions such as the type of HPLC/MS interface and CID parameters. Analytes of which the molecular ion can produce one abundant daughter ion with high yield under CID conditions may be sensitively measured by SRM. ESI is the most soft ionization source developed so far and can afford formation of the fragile molecular ions that are necessary for sensitive SRM detection. Mild CID conditions such as low collision energy and low pressure of collision gas favor production of an abundant daughter ion that is essential to sensitive SRM detection. This knowledge may provide some guidelines for conducting sensitive SRM measurements of very low concentrations of drugs or toxicants in biological samples.     

Disposable hydrophobic surface on MALDI targets for enhancing MS and MS/MS data of peptides

Automated analyses in MALDI MS are complicated by the uneven distribution of analyte over the sample spot, resulting in areas of analyte localization, or "sweet spots". Hence, the ability to concentrate and localize samples is advantageous, especially for automated studies involving low concentrations of analyte. A method for rapidly creating a removable and affordable hydrophobic surface that is free from memory effect is presented. The potential for such compounds to serve as a practical coating for MALDI targets is examined. An example compound with a complete methodology is shown to increase sample homogeneity, peak intensity, and resolution when used for peptide mixtures with CHCA and DHB.     

ZnO-poly(methyl methacrylate) nanobeads for enriching and desalting low-abundant proteins followed by directly MALDI-TOF MS analysis

A fast solid-phase microextraction method using core-shell ZnO-poly (methyl methacrylate) nanobeads (ZnO-PMMA) as adsorbent was established. This fast method with high enriching efficiency and salt tolerance capability depends on the structure of the core-shell nanobeads. First, the large surface area of the PMMA shell makes the dispersive nanobeads capture samples quickly, by virtue of multi-interactions between ZnO-PMMA and samples except for the interaction with salts. Second, the small nanosize of the ZnO-core (2.1 nm) and the flexible hydrophobic PMMA shell, which can prevent the cores from aggregation, make the nanobeads form a homogeneous layer on the matrix-assisted laser desorption/ionization (MALDI) plate and do not hinder the cocrystallization of the matrix and samples. Third, the ZnO core also prevents PMMA from fragmentation and ionization in mass spectrometer. In this article, approximately 80% bovine serum albumin digests were enriched by ZnO-PMMA from 100 amol/muL solution within 10-min incubation, and the solid phase can be directly analyzed by MALDI mass spectrometry. Mass intensity can be increased 5-10-fold (ZnO-PMMA enrichment vs lyophilization). High-quality mass spectra can be obtained, even with the presence of saturated NaCl (6.2 M), saturated NH 4HCO 3 (2.6 M), or 1 M urea. This method has been successfully applied to human colorectal cancer proteome research, and eight new proteins have been found.     

Identification and quantification of neuropeptides in brain tissue by capillary liquid chromatography coupled off-line to MALDI-TOF and MALDI-TOF/TOF-MS

Capillary liquid chromatography (CLC) coupled off-line with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF-MS were explored for identification and quantification of neuropeptides in microwave-fixed rat brain tissue. Sample was separated by gradient elution on 50-mum-inner diameter reversed-phase columns at 180 nL/min. Effluent was mixed with matrix solution and transferred to a MALDI target plate by pulsed electric field deposition, yielding sample spots with 200-300-mum diameter. Mass detection limits as low as 2 amol, corresponding to 1 pM concentration, were achieved for neuropeptides. CLC-MALDI-TOF-MS analysis of microwave-fixed rat striatum tissue yielded detection of over 400 distinctive peaks. CLC-MALDI-TOF/TOF-MS allowed identification of 10 peptides including 3 novel peptides. Quantification was evaluated using substance P as analyte and 15N3-labeled substance P as an internal standard. Quantification of substance P revealed approximately 6.8-fold higher levels than previously reported in the rat striatum. This increase is attributed to use of microwave fixation, which prevented degradation of the peptide, aggressive extraction procedures, and accounting for oxidation of substance P in the analysis. These results demonstrate that CLC-MALDI-TOF-MS is a versatile tool for neuropeptide analysis in brain tissue by allowing for detection, identification, and quantification.     

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.     

Low-attomole electrospray ionization MS and MS/MS analysis of protein tryptic digests using 20-microm-i.d. polystyrene-divinylbenzene monolithic capillary columns

This work explores the use of 20-microm-i.d. polymeric polystyrene-divinylbenzene monolithic nanocapillary columns for the LC-ESI-MS analysis of tryptic digest peptide mixtures. In contrast to the packing of microparticles, capillary columns were prepared, without the need of high pressure, in fused-silica capillaries, by thermally induced in situ copolymerization of styrene and divinylbenzene. The polymerization conditions and mobile-phase composition were optimized for chromatographic performance leading to efficiencies over 100000 plates/m for peptide separations. High mass sensitivity (approximately 10 amol of peptides) in the MS and MS/MS modes using an ion trap MS was found, a factor of up to 20-fold improvement over 75-microm-i.d. nanocolumns. A wide linear dynamic range (approximately 4 orders of magnitude) was achieved, and good run-to-run and column-to-column reproducibility of isocratic and gradient elution separations were found. As samples, both model proteins and tissue extracts were employed. Gradient nano-LC-MS analysis of a proteolytic digest of a tissue extract, equivalent to a sample size of approximately 1000 cells injected, is presented.     

Mass-balanced 1H/2H isotope dipeptide tag for simultaneous protein quantitation and identification

Mass-balanced (1)H/(2)H isotope dipeptide tags (MBITs) are presented for simultaneous protein quantitation and identification. MBIT is derived from N-acetyl-Ala-Ala dipeptide and conjugated to primary amines of target peptides. (1)H/(2)H isotopes are encoded in the methyl groups of N-acetylated dipeptide: one tag deuterated on the N-acetyl group and another on the C-terminal alanine. MBIT-linked peptides comigrate in reversed-phase liquid chromatography without significant (1)H/(2)H isotope effects and provide 2-plex quantitation signals at 114 and 117 Th as well as peptide sequence information upon MS/MS analysis with MALDI TOF/TOF. MBIT shows good quantitation linearity in a concentration range of 20-250 fmol. The performance of MBIT on protein quantitation and identification is further tested with yeast heat-shock protein (Hsp82p) obtained from three different physiological states. MBIT using nanogram-scale samples produces the relative abundance ratios comparable to those obtained from optical imaging of microgram-scale samples visualized with SYPRO Ruby stain. The MBIT strategy is a simple and low-cost alternative for 2-plex quantitation of proteins and offers possibilities of tuning the 2-plex signal mass window by replacing the N-terminal alanine with other amino acid residues.     

Fluorescence-based peptide labeling and fractionation strategies for analysis of cysteine-containing peptides

This study demonstrates that 1,5-I-AEDANS (5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid) can be used as a versatile fluorescence-based peptide quantification tool and provides readily interpretable tandem mass spectra for de novo peptide sequencing. Two AEDANS-cysteinyl-peptide fractionation strategies were evaluated. One AEDANS-cysteinyl-peptide fractionation strategy employs immobilized metal affinity chromatography (IMAC) to recover AEDANS-labeled peptides and reduce the complexity of peptide mixtures. In an alternate solid-phase approach, 1,5-I-AEDANS was coupled to an o-nitrobenzyl-based photocleavable resin to produce a resin that can label and isolate thiols and cysteine-containing peptides with a modified-AEDANS label (mAEDANS: 5-((4-amino-4-oxobutanoyl){2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid). This fractionation protocol enriches cysteine-containing peptides more specifically than the IMAC strategy. Using micro-LC-ESI-MS with an on-line fluorescence detector and a Q-TOF mass spectrometer, we generated fluorescence-based elution profiles and corresponding positive ion mass spectra of AEDANS-labeled peptides. This study demonstrates that AEDANS-peptides produce positive ion ESI-MS mass spectra with detection limits comparable to those of the unlabeled peptide. Collision-induced dissociation (CID) of fluorescent AEDANS-peptides revealed readily interpretable product ion spectra with the label intact. Similar to the AEDANS-labeled peptide, an mAEDANS-labeled thiol is fluorescent and CID of a mAEDANS-labeled peptide also reveals an interpretable product ion spectrum with the label intact.