Differential dimethyl labeling of N-termini of peptides after guanidination for proteome analysis

We describe an enabling technique for proteome analysis based on isotope-differential dimethyl labeling of N-termini of tryptic peptides followed by microbore liquid chromatography (LC) matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS). In this method, lysine side chains are blocked by guanidination to prevent the incorporation of multiple labels, followed by N-terminal labeling via reductive amination using d(0),(12)C-formaldehyde or d(2),(13)C-formaldehyde. Relative quantification of peptide mixtures is achieved by examining the MALDI mass spectra of the peptide pairs labeled with different isotope tags. A nominal mass difference of 6 Da between the peptide pair allows negligible interference between the two isotopic clusters for quantification of peptides of up to 3000 Da. Since only the N-termini of tryptic peptides are differentially labeled and the a(1) ions are also enhanced in the MALDI MS/MS spectra, interpretation of the fragment ion spectra to obtain sequence information is greatly simplified. It is demonstrated that this technique of N-terminal dimethylation (2ME) after lysine guanidination (GA) or 2MEGA offers several desirable features, including simple experimental procedure, stable products, using inexpensive and commercially available reagents, and negligible isotope effect on reversed-phase separation. LC-MALDI MS combined with this 2MEGA labeling technique was successfully used to identify proteins that included polymorphic variants and low abundance proteins in bovine milk. In addition, by analyzing a mixture of two equal amounts of milk whey fraction as a control, it is shown that the measured average ratio for 56 peptide pairs from 14 different proteins is 1.02, which is very close to the theoretical ratio of 1.00. The calculated percentage error is 2.0% and relative standard deviation is 4.6%.     

Sequence tag scanning: a new explorative strategy for recognition of unexpected protein alterations by nanoelectrospray ionization-tandem mass spectrometry

Protein analysis by database search engines using tandem mass spectra is limited by the presence of unexpected protein modifications, sequence isoforms which may not be in the protein databases, and poor quality tandem mass spectrometry (MS/MS) of low abundance proteins. The analysis of expected protein modifications can be efficiently addressed by precursor ion scanning. However, it is limited to modifications that show such a characteristic loss in a peptide independent manner. We observed that proline and aspartic acid induced backbone fragmentation is accompanied by a low intensity signal for loss of H3PO4 for several pSer- or pThr-phosphopeptides. We describe here the use of peptide-specific fragments that can be used after a protein was identified to allow in-depth characterization of modifications and isoforms. We consider high abundance fragments formed by cleavage at the C-terminal side of aspartic acid, at the N-terminal side of proline and low mass ions such as a2, b2, b3, y1, y2, and y3. The MS/MS dataset is filtered for each sequence tag of interest by an in silico precursor ion scan. The resulting extracted ion traces are then combined by multiplication to increase specificity. Since the strategy is based on common peptide segments which are shared by different isoforms of peptides it can be applied to the analysis of any post-translational modification or sequence variants of a protein. This is demonstrated for the cases of serine and threonine phosphorylation, histone H1 acetylation and the spotting of multiple H1 isoforms.     

Highly informative proteome analysis by combining improved N-terminal sulfonation for de novo peptide sequencing and online capillary reverse-phase liquid chromatography/tandem mass spectrometry

Recently, various chemical modifications of peptides have been incorporated into mass spectrometric analyses of proteome samples, predominantly in conjunction with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), to facilitate de novo sequencing of peptides. In this work, we investigate systematically the utility of N-terminal sulfonation of tryptic peptides by 4-sulfophenyl isothiocyanate (SPITC) for proteome analysis by capillary reverse-phase liquid chromatography/tandem mass spectrometry (cRPLC/MS/MS). The experimental conditions for the sulfonation were carefully adjusted so that SPITC reacts selectively with the N-terminal amino groups, even in the presence of the epsilon-amino groups of lysine residues. Mass spectrometric analyses of the modified peptides by cRPLC/MS/MS indicated that SPITC derivatization proceeded toward near completion under the experimental conditions employed here. The SPITC-derivatized peptides underwent facile fragmentation, predominantly resulting in y-series ions in the MS/MS spectra. Combining SPITC derivatization and cRPLC/MS/MS analyses facilitated the acquisition of sequence information for lysine-terminated tryptic peptides as well as arginine-terminated peptides without the need for additional peptide pretreatment, such as guanidination of lysine amino group. This process alleviated the biased detection of arginine-terminated peptides that is often observed in MALDI MS experiments. We will discuss the utility of the technique as a viable method for proteome analyses and present examples of its application in analyzing samples having different levels of complexity.     

Mass spectrometric strategy for primary structure determination of N-terminally blocked peptides

The mass spectrometric strategy including three steps is presented for primary structure determination of the N-terminally blocked peptides. First, the C-terminal sequencing is performed by using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry coupled with carboxypeptidase Y digestion. Then, the peptide is cleaved according to the obtained C-terminal sequence information and the resulting peptides are identified by mass spectrometry and Edman degradation after fractionation by reverse-phase chromatography. Finally, the N-terminal fragment is sequenced by tandem mass spectrometry. The strategy was successfully applied to the sequence determination of two novel N-terminally blocked peptides named EAFP1 and EAFP2.     

Trypsin cleaves exclusively C-terminal to arginine and lysine residues

Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments and show that trypsin cleaves solely C-terminal to arginine and lysine. We find that non-tryptic peptides occur only as the C-terminal peptides of proteins and as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins erroneously identified with non-tryptic peptide hits. Our results indicate that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity.     

Patchwork peptide sequencing: extraction of sequence information from accurate mass data of peptide tandem mass spectra recorded at high resolution

The accurate mass values of all immonium, y(1), y(2), a(2), and b(2) ions of tryptic peptides composed of the 20 standard amino acids were calculated. The differences between adjacent masses in this data set are greater than 10 mDa for more than 80% of the values. Using this mass list, the majority of low mass ions in quadrupole-time of flight tandem mass spectra of peptides from tryptic digests and from an elastase digest could be assigned. Besides the a(2)/b(2) ions, which carry residues 1-2 from the N-terminus, a variety of internal dipeptide b ions were regularly observed. In case internal proline was present, corresponding dipeptide b ions carrying proline at the N-terminal position occurred. By assigning the dipeptide b ions on the basis of their accurate mass, bidirectional or unidirectional sequence information was obtained, which is localized to the peptide N-terminus (a(2)/b(2) ions) or not localized (internal b ions). Identification of the y(1) and y(2) ions by their accurate mass provides unidirectional sequence information localized to the peptide C-terminus. It is shown that this patchwork-type sequence information extractable from accurate mass data of low-mass ions is highly efficient for protein identification.     

Increased protein identification capabilities through novel tandem MS calibration strategies

High mass measurement accuracy is critical for confident protein identification and characterization in proteomics research. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is a unique technique which can provide unparalleled mass accuracy and resolving power. However, the mass measurement accuracy of FTICR-MS can be affected by space charge effects. Here, we present a novel internal calibrant-free calibration method that corrects for space charge-induced frequency shifts in FTICR fragment spectra called Calibration Optimization on Fragment Ions (COFI). This new strategy utilizes the information from fixed mass differences between two neighboring peptide fragment ions (such as y(1) and y(2)) to correct the frequency shift after data collection. COFI has been successfully applied to LC-FTICR fragmentation data. Mascot MS/MS ion search data demonstrate that most of the fragments from BSA tryptic digested peptides can be identified using a much lower mass tolerance window after applying COFI to LC-FTICR-MS/MS of BSA tryptic digest. Furthermore, COFI has been used for multiplexed LC-CID-FTICR-MS which is an attractive technique because of its increased duty cycle and dynamic range. After the application of COFI to a multiplexed LC-CID-FTICR-MS of BSA tryptic digest, we achieved an average measured mass accuracy of 2.49 ppm for all the identified BSA fragments.     

Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.     

Laser-induced dissociation of phosphorylated peptides using matrix assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Reversible phosphorylation is one of the most important posttranslational modifications of cellular proteins. Mass spectrometry is a widely used technique in the characterization of phosphorylated proteins and peptides. Similar to nonmodified peptides, sequence information for phosphopeptides digested from proteins can be obtained by tandem mass analysis using either electrospray ionization or matrix assisted laser desorption/ionization (MALDI) mass spectrometry. However, the facile loss of neutral phosphoric acid (H₃PO₄) or HPO₃ from precursor ions and fragment ions hampers the precise determination of phosphorylation site, particularly if more than one potential phosphorylation site or concensus sequence is present in a given tryptic peptide. Here, we investigated the fragmentation of phosphorylated peptides under laser-induced dissociation (LID) using a MALDI-time-of-flight mass spectrometer with a curved-field reflectron. Our data demonstrated that intact fragments bearing phosphorylated residues were produced from all tested peptides that contain at least one and up to four phosphorylation sites at serine, threonine, or tyrosine residues. In addition, the LID of phosphopeptides derivatized by N-terminal sulfonation yields simplified MS/MS spectra, suggesting the combination of these two types of spectra could provide an effective approach to the characterization of proteins modified by phosphorylation.     

Identification of a novel phosphorylation site in human androgen receptor by mass spectrometry

An N-terminal hexahistidine-tagged full-length human androgen receptor protein (His(6)-hAR) was overexpressed and purified to apparent homogeneity in the presence of dihydrotestosterone (DHT) in our previous studies. In-gel trypsin digestion of the purified DHT-bound His(6)-hAR, and tryptic peptide mapping using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), detected a total of 17 peptides (21% coverage of hAR) with 9 peptides originating from the ligand-binding domain (LBD, 31% coverage of LBD). Amino acid sequencing analysis of the tryptic peptides from a separate in-gel digestion of the His(6)-hAR, using HPLC-coupled electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS and MS/MS), unambiguously confirmed 21 peptides with 19% coverage of the hAR, of which 11 peptides originated from the LBD (35% coverage of LBD). These 21 peptides included 11 out of the 17 peptides detected by MALDI/TOF-MS. In addition, a novel serine phosphorylation site (Ser(308)) within the N-terminal transactivation domain of hAR was identified.     

Mass spectrometric characterization of the human androgen receptor ligand-binding domain expressed in Escherichia coli

The ligand-binding domain (LBD) of the human androgen receptor (hAR LBD), encompassing amino acids (AAs) 647-919, was expressed in Escherichia coli with an N-terminal polyhistidine tag (His(10)-hAR LBD) from a pET-16b vector. The overexpressed protein was initially insoluble in inclusion bodies, and was subsequently solubilized in 8 M guanidine hydrochloride (GdnHCl). The solubilized His(10)-hAR LBD was purified to apparent homogeneity by metal ion affinity chromatography in the presence of 6 M GdnHCl. The isolated protein migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of 33-34 kDa, as expected from the plasmid construct. Immunoblot analysis with C-terminal antibodies raised against a peptide corresponding to the last 19 AAs (AAs 901-919) of hAR revealed that the purified protein contained an immunoreactive epitope present within the AR and was of the appropriate size. Further characterization, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), showed a single protein species of average mass 34 580 Da, confirming the size and purity of the purified His(10)-hAR LBD. Detailed tryptic peptide mapping analysis, using MALDI/TOF-MS, identified a total of eight peptides with a 30% coverage of the LBD, including the last tryptic peptide in the hAR sequence. These data confirm that the purified protein was the intact hAR LBD. AA sequencing of these tryptic peptides, using an HPLC-coupled electrospray ionization ion trap mass spectrometer (LC/ESI-ITMS and MS/MS), unambiguously confirmed that the peptides were from the hAR LBD. The purified His(10)-hAR LBD in 6 M GdnHCl could be renatured as determined by ligand-binding activity, with a similar equilibrium dissociation constant (K(d)) for [(3)H]-mibolerone and a similar steroid specificity to the AR isolated from rat ventral prostate.     

Matrix-assisted laser desorption/ionization mass spectrometry peptide sequencing utilizing selective N-terminal bromoacetylation

In tandem mass spectrometric peptide sequencing, simplifying the mass spectrum is often desirable. The b-series ions were distinguished from the y-series ions in the MALDI TOF-TOF spectra by incorporating a bromine-tag to the N-terminal amino group through rapid and selective acetylation using bromoacetic anhydride without blocking the lysine and tyrosine residues. The 51:49 ratio of Br-79 and Br-81 isotopes facilitated identification of ions carrying the tag. With the Br-tag in the b-series ions, N-terminal sequencing of tryptic peptides from hemoglobin as well as model peptides was straightforward. When the b-ions were low in intensity, ions without the Br-tag were identified as y-ions and used for sequencing.     

Experimental protein mixture for validating tandem mass spectral analysis

Several methods have been used to identify peptides that correspond to tandem mass spectra. In this work, we describe a data set of low energy tandem mass spectra generated from a control mixture of known protein components that can be used to evaluate the accuracy of these methods. As an example, these spectra were searched by the SEQUEST application against a human peptide sequence database. The numbers of resulting correct and incorrect peptide assignments were then determined. We show how the sensitivity and error rate are affected by the use of various filtering criteria based upon SEQUEST scores and the number of tryptic termini of assigned peptides.     

A suboptimal algorithm for de novo peptide sequencing via tandem mass spectrometry.

Tandem mass spectrometry has emerged to be one of the most powerful high-throughput techniques for protein identification. Tandem mass spectrometry selects and fragments peptides of interest into N-terminal ions and C-terminal ions, and it measures the mass/charge ratios of these ions. The de novo peptide sequencing problem is to derive the peptide sequences from given tandem mass spectral data of k ion peaks without searching against protein databases. By transforming the spectral data into a matrix spectrum graph G = (V, E), where |V| = O(k(2)) and |E| = O(k(3)), we give the first polynomial time suboptimal algorithm that finds all the suboptimal solutions (peptides) in O(p|E|) time, where p is the number of solutions. The algorithm has been implemented and tested on experimental data. The program is available at http://hto-c.usc.edu:8000/msms/menu/denovo.htm.     

The fate of b-ions in the two worlds of collision-induced dissociation

Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16% of b-ion loss.     

Identification of transmembrane tryptic peptides of rhodopsin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The application of mass spectrometry for determining the topography of integral membrane proteins has focused primarily on the mass determination of fragments that do not reside in the lipid bilayer. In this work, we present the accurate mass determination of transmembrane tryptic peptides of bovine rhodopsin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The ability to determine the accurate mass of hydrophobic transmembrane peptides will facilitate the mapping of ligand binding sites in membrane receptors. It will also augment the determination of membrane spanning regions from integral membrane proteins digested in lipid bilayers. Affinity-purified rhodopsin in detergent and rhodopsin in retinal rod membranes were digested with trypsin. Tryptic peptides were separated using reverse-phase, high-performance liquid chromatography at 55 degrees C with the detergent octyl-beta-glucoside in the mobile phase. Four of the six transmembrane tryptic peptides of rhodopsin were identified, ranging in mass from 3,260 Da to 6,528 Da. The identities of the peptides were confirmed by Edman microsequencing. In addition, heterogeneity in the glycosylation of the N-terminal tryptic peptide of rhodopsin was identified by MALDI MS, without modifying the carbohydrate prior to analysis.     

Mass-Spectrometry Based Characterisation of Infant Whole Saliva Peptidome

The objective of the study was to determine optimal conditions for sampling, sample processing and mass-spectrometry based analysis of infants’ salivary peptidome. Saliva was sampled in 3- and 6-month-old infants and peptide extracts were prepared. Various sample pretreatments before profiling by MALDI-ToF were evaluated and peptide identification was undertaken by MALDI-ToF/ToF or nanoLC-ESI-IT tandem MS. A fast and simple protocol (cut-off filtration at 5 kDa) was satisfactory to produce extracts where no proteolysis was detected even when no protease inhibitor was added. Optimal MALDI spectra were generated after purification on C18 tips. Variability of spectra between two samples exceeded that of the technical replicates, validating that the method is suitable to conduct differential studies. Salivary peptides, identified by means of the two complementary mass spectrometry techniques, were fragments of proline-rich proteins and histatins. The fragments originated mainly from the C-terminal protein extremities. Indications on the proteolytic systems involved and the anatomic location where they intervene are proposed.     

Integrated approach for manual evaluation of peptides identified by searching protein sequence databases with tandem mass spectra

Quantitative proteomics relies on accurate protein identification, which often is carried out by automated searching of a sequence database with tandem mass spectra of peptides. When these spectra contain limited information, automated searches may lead to incorrect peptide identifications. It is therefore necessary to validate the identifications by careful manual inspection of the mass spectra. Not only is this task time-consuming, but the reliability of the validation varies with the experience of the analyst. Here, we report a systematic approach to evaluating peptide identifications made by automated search algorithms. The method is based on the principle that the candidate peptide sequence should adequately explain the observed fragment ions. Also, the mass errors of neighboring fragments should be similar. To evaluate our method, we studied tandem mass spectra obtained from tryptic digests of E. coli and HeLa cells. Candidate peptides were identified with the automated search engine Mascot and subjected to the manual validation method. The method found correct peptide identifications that were given low Mascot scores (e.g., 20-25) and incorrect peptide identifications that were given high Mascot scores (e.g., 40-50). The method comprehensively detected false results from searches designed to produce incorrect identifications. Comparison of the tandem mass spectra of synthetic candidate peptides to the spectra obtained from the complex peptide mixtures confirmed the accuracy of the evaluation method. Thus, the evaluation approach described here could help boost the accuracy of protein identification, increase number of peptides identified, and provide a step toward developing a more accurate next-generation algorithm for protein identification.     

Incorporation of tandem mass spectrometric detection to the analysis of peptide mixtures by continuous flow fast atom bombardment mass spectrometry

The ability to acquire structurally informative daughter ion spectra for individual peptides undergoing separation and analysis by continuous flow fast atom bombardment (CF FAB) is demonstrated. To illustrate the potential of this methodology, tryptic and chymotryptic digests of the 29-residue peptide glucagon were analyzed by CF FAB using mass spectrometric and tandem mass spectrometric detection in consecutive analyses. Daughter ion spectra were recorded using B/E linked scans for the major hydrolysis products observed by liquid chromatography/mass spectrometry. The peptide mixtures were separated by gradient capillary high-performance liquid chromatography with the FAB matrix being added post-column using a coaxial flow interface between the column and flow probe. The entire effluent (3 microl min(-1)) was sampled by the mass spectrometer. Results obtained using less than 300 pmol of digested glucagon indicated several advantages to tandem mass spectrometric detection including the ability to confirm identities for products of enzymatic digestion and the potential use of this method for tandem sequence analysis of peptide mixtures.     

iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer

For proteomic analysis using tandem mass spectrometry, linear ion trap instruments provide unsurpassed sensitivity but unreliably detect low mass peptide fragments, precluding their use with iTRAQ reagent-labeled samples. Although the popular LTQ linear ion trap supports analyzing iTRAQ reagent-labeled peptides via pulsed Q dissociation, PQD, its effectiveness remains questionable. Using a standard mixture, we found careful tuning of relative collision energy necessary for fragmenting iTRAQ reagent-labeled peptides, and increasing microscan acquisition and repeat count improves quantification but identifies somewhat fewer peptides. We developed software to calculate abundance ratios via summing reporter ion intensities across spectra matching to each protein, thereby providing maximized accuracy. Testing found that results closely corresponded between analysis using optimized LTQ-PQD settings plus our software and using a Qstar instrument. Thus, we demonstrate the effectiveness of LTQ-PQD analyzing iTRAQ reagent-labeled peptides, and provide guidelines for successful quantitative proteomic studies.