Sequence dependent fragmentation of peptides generated by MALDI quadrupole time-of-flight (MALDI Q-TOF) mass spectrometry and its implications for protein identification

A study has been undertaken to evaluate the usefulness of MALDI Q-TOF data for protein identification. The comparison of MS data of protein digests obtained on a conventional MALDI TOF instrument to the MS data from the MALDI Q-TOF reveal peptide patterns with similar intensity ratios. However, comparison of MS/MS Q-TOF data produced by nanoelectrospray versus MALDI reveals striking differences. Peptide fragment ions obtained from doubly charged precursors produced by nanoelectrospray are mainly y-type ions with some b-ions in the lower mass range. In contrast, peptide fragment ions produced from the singly charged ions originating from the MALDI source are a mixture of y-, b- and a-ions accompanied by ions resulting from neutral loss of ammonia or water. The ratio and intensity of these fragment ions is found to be strongly sequence dependent for MALDI generated ions. The singly charged peptides generated by MALDI show a preferential cleavage of the C-terminal bond of acidic residues aspartic and glutamic acid and the N-terminal bond of proline. This preferential cleavage can be explained by the mobile proton model and is present in peptides that contain both arginine and an acidic amino acid. The MALDI Q-TOF MS/MS data of 24 out of 26 proteolytic peptides produced by trypsin or Asp-N digestions were successfully used for protein identification via database searching, thus indicating the general usefulness of the data for protein identification. De novo sequencing using a mixture of 160/18O water during digestion has been explored and de novo sequences for a number of peptides have been obtained.     

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.     

磺基异硫氰酸苯酯化学辅助方法对新蛋白质进行从头测序

利用基质辅助激光解吸电离-串联飞行时间(MALDI-TOF-TOF)质谱结合磺基异硫氰酸苯酯化学辅助的方法对一种从拟青霉(Paecilomyces bainier)分离纯化到的新人参皂苷Rb1水解酶的部分肽段进行了从头测序. 共获得了这个新蛋白质8条肽段的序列, 一些磺化后信噪比非常低的肽段也获得了比较完整的序列. 同时通过从头测序分析确定了一对甲硫氨酸非氧化和氧化肽段的序列. 结果表明, 磺化后的肽段离子化效率大大增强, 在PSD(源后裂解)过程中只有肽键断裂产生的C端的碎片离子系列(y离子系列)出现在质谱图中, 图谱背景清晰, 信噪比高, 单纯的y离子系列使得图谱解析变得非常容易. 将这8条序列在NCBI(美国国立生物技术信息中心)数据库中进行BLAST(蛋白质序列比对工具)检索印证这种β-葡萄糖甘酶是一个新蛋白质, 发现的两条相对保守的序列为进一步研究奠定了基础.     

CHASE, a charge-assisted sequencing algorithm for automated homology-based protein identifications with matrix-assisted laser desorption/ionization time-of-flight post-source decay fragmentation data

We describe CHASE, a novel algorithm for automated de novo sequencing based on the mass spectrometric (MS) fragmentation analysis of tryptic peptides. This algorithm is used for protein identification from sequence similarity criteria and consists of four steps: (1) derivatization of tryptic peptides at the N-terminus with a negatively charged reagent; (2) post-source decay (PSD) fragmentation analysis of peptides; (3) interpretation of the mass peaks with the CHASE algorithm and reconstruction of the amino acid sequence; (4) transfer of these data to software for protein identifications based on sequence homology (Basic Local Alignment Search Tool, BLAST). This procedure deduced the correct amino acid sequence of tryptic peptide samples and also was able to deduce the correct sequence from difficult mass patterns and identify the amino acid sequence. This allows complete automation of the process starting from MS fragmentation of complex peptide mixtures at low concentration (e.g. from silver-stained gel bands) to identification of the protein. We also show that if PSD data are collected in a single spectrum (instead of the segmented mode offered by conventional matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation), the complete workflow from MS-PSD data acquisition to similarity-based identification can be completely automated. This strategy may be applied to proteomic studies for protein identification based on automated de novo sequencing instead of MS or tandem MS patterns. We describe the Charge Assisted Sequencing Engine (CHASE) algorithm, the working protocol, the performance of the algorithm on spectra from MALDI-TOFMS and the data comparison between a TOF and a TOF-TOF instrument.     

'Information-Based-Acquisition' (IBA) technique with an ion-trap/time-of-flight mass spectrometer for high-throughput and reliable protein profiling

Highly complex protein mixtures can be analyzed after proteolysis using liquid chromatography/mass spectrometry (LC/MS). In an LC/MS run, intense peptide ions originating from high-abundance proteins are preferentially analyzed using tandem mass spectrometry (MS(2)), so obtaining the MS(2) spectra of peptide ions from low-abundance proteins is difficult even if such ions are detected. Furthermore, the MS(2) spectra may produce insufficient information to identify the peptides or proteins. To solve these problems, we have developed a real-time optimization technique for MS(2), called the Information-Based-Acquisition (IBA) system. In a preliminary LC/MS run, a few of the most intense ions detected in every MS spectrum are selected as precursors for MS(2) and their masses, charge states and retention times are automatically registered in an internal database. In the next run, a sample similar to that used in the first run is analyzed using database searching. Then, the ions registered in the database are excluded from the precursor ion selection to avoid duplicate MS(2) analyses. Furthermore, real-time de novo sequencing is performed just after obtaining the MS(2) spectrum, and an MS(3) spectrum is obtained for accurate peptide identification when the number of interpreted amino acids in the MS(2) spectrum is less than five. We applied the IBA system to a yeast cell lysate which is a typical crude sample, using a nanoLC/ion-trap time-of flight (IT/TOF) mass spectrometer, repeating the same LC/MS run five times. The obtained MS(2) and MS(3) spectra were analyzed by applying the Mascot (Matrix Science, Boston, MA, USA) search engine to identify proteins from the sequence database. The total number of identified proteins in five LC/MS runs was three times higher than that in the first run and the ion scores for peptide identification also significantly increased, by about 70%, when the MS(3) spectra were used, combined with the MS(2) spectra, before being subjected to Mascot analysis.     

Chimera Spectrum Diagnostics for Peptides Using Two-Dimensional Partial Covariance Mass Spectrometry

The rate of successful identification of peptide sequences by tandem mass spectrometry (MS/MS) is adversely affected by the common occurrence of co-isolation and co-fragmentation of two or more isobaric or isomeric parent ions. This results in so-called `chimera spectra’, which feature peaks of the fragment ions from more than a single precursor ion. The totality of the fragment ion peaks in chimera spectra cannot be assigned to a single peptide sequence, which contradicts a fundamental assumption of the standard automated MS/MS spectra analysis tools, such as protein database search engines. This calls for a diagnostic method able to identify chimera spectra to single out the cases where this assumption is not valid. Here, we demonstrate that, within the recently developed two-dimensional partial covariance mass spectrometry (2D-PC-MS), it is possible to reliably identify chimera spectra directly from the two-dimensional fragment ion spectrum, irrespective of whether the co-isolated peptide ions are isobaric up to a finite mass accuracy or isomeric. We introduce ‘3-57 chimera tag’ technique for chimera spectrum diagnostics based on 2D-PC-MS and perform numerical simulations to examine its efficiency. We experimentally demonstrate the detection of a mixture of two isomeric parent ions, even under conditions when one isomeric peptide is at one five-hundredth of the molar concentration of the second isomer.     

Improved protein identification efficiency by mass spectrometry using N-terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome

Matrix-assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI-MS/MS ion search (using MASCOT) have become the preferred methods for high-throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post-source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI-TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanate (SPITC) prior to MALDI-TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI-TOF/TOF-PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules.     

Improved protein identification efficiency by mass spectrometry using N-terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome

Matrix-assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI-MS/MS ion search (using MASCOT) have become the preferred methods for high-throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post-source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI-TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanate (SPITC) prior to MALDI-TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI-TOF/TOF-PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules.     

"De novo" peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: a preliminary study

Collision-induced dissociation of singly charged peptide ions produced by resonant excitation in a matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer yields relatively low complexity MS/MS spectra that exhibit highly preferential fragmentation, typically occurring adjacent to aspartyl, glutamyl, and prolyl residues. Although these spectra have proven to be of considerable utility for database-driven protein identification, they have generally been considered to contain insufficient information to be useful for extensive de novo sequencing. Here, we report a procedure for de novo sequencing of peptides that uses MS/MS data generated by an in-house assembled MALDI-quadrupole-ion trap mass spectrometer (Krutchinsky, Kalkum, and Chait Anal. Chem. 2001, 73, 5066-5077). Peptide sequences of up 14 amino acid residues in length have been deduced from digests of proteins separated by SDS-PAGE. Key to the success of the current procedure is an ability to obtain MS/MS spectra with high signal-to-noise ratios and to efficiently detect relatively low abundance fragment ions that result from the less favorable fragmentation pathways. The high signal-to-noise ratio yields sufficiently accurate mass differences to allow unambiguous amino acid sequence assignments (with a few exceptions), and the efficient detection of low abundance fragment ions allows continuous reads through moderately long stretches of sequence. Finally, we show how the aforementioned preferential cleavage property of singly charged ions can be used to facilitate the de novo sequencing process.     

Improved peptide sequencing using isotope information inherent in tandem mass spectra

We demonstrate here the use of natural isotopic 'labels' in peptides to aid in the identification of peptides with a de novo algorithm. Using data from ion trap tandem mass spectrometric (MS/MS) analysis of 102 tryptic peptides, we have analyzed multiple series of peaks within LCQ MS/MS spectra that 'spell' peptide sequences. Isotopic peaks from naturally abundant isotopes are particularly prominent even after peak centroiding on y- and b-series ions and lead to increased confidence in the identification of the precursor peptides. Sequence analysis of the MS/MS data is accomplished by finding sequences and subsequences in a hierarchical manner within the spectra.     

Automated interpretation of low-energy collision-induced dissociation spectra by SeqMS, a software aid for de novo sequencing by tandem mass spectrometry

SeqMS, a software aid for de novo sequencing by tandem mass spectrometry (MS/MS), which was initially developed for the automated interpretation of high-energy collision-induced dissociation (CID) MS/MS spectra of peptides, has been applied to the interpretation of low-energy CID and post-source decay (PSD) spectra of peptides. Based on peptide backbone fragmented ions and their related ions, which are the dominant ions observed in the latter two techniques, the types of ions and their propensities to be observed have been optimized for efficient interpretation of the spectra. In a typical example, the modified SeqMS allowed the complete sequencing of a 31-amino acid synthetic peptide, except for the isobaric amino acids (Leu or Ile, and Lys or Gln), based on only the low-energy CID-MS/MS spectrum.     

De novo sequencing,peptide composition analysis,and composition-based sequencing: A new strategy employing accurate mass determination by fourier transform ion cyclotron resonance mass spectrometry

A new strategy is described for the determination of amino acid sequences of unknown peptides. Different from the well-known but often inefficient de novo sequencing approach, the new method is based on a two-step process. In the first step the amino acid composition of an unknown peptide is determined on the basis of accurate mass values of the peptide precursor ion and a small number of accurate fragment ion mass values, and, as in de novo sequencing, without employing protein database information or other pre-information. In the second step the sequence of the found amino acids of the peptide is determined by scoring the agreement between expected and observed fragment ion signals of the permuted sequences. It was found that the new approach is highly efficient if accurate mass values are available and that it easily outstrips common approaches of de novo sequencing being based on lower accuracies and detailed knowledge of fragmentation behavior. Simple permutation and calculation of all possible amino acid sequences, however, is only efficient if the composition is known or if possible compositions are at least reduced to a small list. The latter requires the highest possible instrumental mass accuracy, which is currently provided only by fourier transform ion cyclotron resonance mass spectrometry. The connection between mass accuracy and peptide composition variability is described and an example of peptide compositioning and composition-based sequencing is presented.     

De novo analysis of protein N-terminal sequence utilizing MALDI signal enhancing derivatization with Br signature

De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.     

How much peptide sequence information is contained in ion trap tandem mass spectra?

Matching peptide tandem mass spectra to their cognate amino acid sequences in databases is a key step in proteomics. It is usually performed by assigning a score to a spectrum-sequence combination. De novo sequencing or partial de novo sequencing is useful for organisms without sequenced genome or for peptides with unexpected modifications. Here we use a very large, high accuracy proteomic dataset to investigate how much peptide sequence information is present in tandem mass spectra generated in a linear ion trap (LTQ). More than 400,000 identified tandem mass spectra from a single human cancer cell line project were assigned to 26,896 distinct peptide sequences. The average absolute fragment mass accuracy is 0.102 Da. There are on average about four complementary b- and y-ions; both series are equally represented but y ions are 2- to 3-fold more intense up to mass 1000. Half of all spectra contain uninterrupted b- or y-ion series of at least six amino acids and combining b- and y-ion information yields on average seven amino acid sequences. These sequences are almost always unique in the human proteome, even without using any precursor or peptide sequence tag information. Thus, optimal de novo sequencing algorithms should be able to obtain substantial sequence information in at least half of all cases.     

Characterization and de novo sequencing of Atlantic salmon vitellogenin protein by electrospray tandem and matrix-assisted laser desorption/ionization mass spectrometry

Vitellogenin (VTG) is a protein produced by the liver of oviparous animals in response to circulating estrogens. In the plasma of males and immature females, VTG is undetectable. VTG has been used as a biomarker for exposure to endocrine disruptors in many species. In the present study, characterization of intact Atlantic salmon VTG was effected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). Tryptic digest peptides were analyzed by MALDI ToF MS to obtain a peptide mass fingerprint. De novo sequencing of the tryptic peptides used low-energy collisionally-induced dissociation (CID) in an electrospray ionization quadrupole-ToF orthogonal hybrid mass spectrometer (ESI Q-ToF MS/MS). The interpretation of the product-ion spectra obtained from the ESI Q-ToF MS/MS was done by Lutefisk, a computer-based software algorithm. The molecular mass of the intact protein was found to be 187335 Da. A total of 14 tryptic peptides were sequenced and compared with the complete rainbow trout VTG and the partial Atlantic salmon VTG sequences found in the Swiss-Prot database. De novo sequencing by CID MS/MS of 11 Atlantic salmon tryptic digest peptides with selected precursor ions at m/z 788.24, 700.20, 794.75, 834.31, 889.28, 819.79, 865.27, 843.81, 572.20, 573.66 and 561.68 showed high homology with the known sequence of rainbow trout VTG. The last two precursor peptide ions, found at m/z 573.66 and m/z 561.68, also specifically matched the known portion of the Atlantic salmon VTG sequence. Finally, three tryptic precursor peptide ions found at m/z 795.18, 893.28 and 791.05, provided product-ion spectra, which were exclusive to the unsequenced portion of the Atlantic salmon VTG.     

On-line capillary liquid chromatography tandem mass spectrometry on an ion trap/ reflectron time-of-flight mass spectrometer using the sequence tag database search approach for peptide sequencing and protein identification

Capillary high-performance liquid chromatography has been coupled on-line with an ion trap storage/reflectron time-of-flight mass spectrometer to perform tandem mass spectrometry for tryptic peptides. Selection and fragmentation of the precursor ions were performed in a three-dimensional ion trap, and the resulting fragment ions were pulsed out of the trap into a reflectron time-of-flight mass spectrometer for mass analysis. The stored waveform inverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collision-induced dissociation. Tandem mass spectra of various doubly charged tryptic peptides were investigated where a conspicuous y ion series over a certain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by database search using the sequence tag approach. Several peptides from tryptic digests of horse heart myoglobin and bovine cytochrome c were selected for tandem mass spectrometry (MS/MS) where it was demonstrated that the proteins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimensional gel separation of a human esophageal adenocarcinoma cell line.     

A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics

A new matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometer with the novel "LIFT" technique (MALDI LIFT-TOF/TOF MS) is described. This instrument provides high sensitivity (attomole range) for peptide mass fingerprints (PMF). It is also possible to analyze fragment ions generated by any one of three different modes of dissociation: laser-induced dissociation (LID) and high-energy collision-induced dissociation (CID) as real MS/MS techniques and in-source decay in the reflector mode of the mass analyzer (reISD) as a pseudo-MS/MS technique. Fully automated operation including spot picking from 2D gels, in-gel digestion, sample preparation on MALDI plates with hydrophilic/hydrophobic spot profiles and spectrum acquisition/processing lead to an identification rate of 66% after the PMF was obtained. The workflow control software subsequently triggered automated acquisition of multiple MS/MS spectra. This information, combined with the PMF increased the identification rate to 77%, thus providing data that allowed protein modifications and sequence errors in the protein sequence database to be detected. The quality of the MS/MS data allowed for automated de novo sequencing and protein identification based on homology searching.     

Automated de novo sequencing of nucleic acids by liquid chromatography-tandem mass spectrometry

We present the first global computer-aided sequencing algorithm for the de novo determination of short nucleic acid sequences. The method compares the fragment ion spectra generated by collision-induced dissociation of multiply charged oligodeoxynucleotide-ions to the m/z values predicted employing established fragmentation pathways from a known reference sequence. The closeness of matching between the measured spectrum and the predicted set of fragment ions is characterized by the fitness, which takes into account the difference between measured and predicted m/z values, the intensity of the fragment ions, the number of fragments assigned, and the number of nucleotide positions not covered by fragment ions in the experimental spectrum. Smaller values for the fitness indicate a closer match between the measured spectrum and predicted m/z values. In order to find the sequence most closely matching the experimental spectrum, starting from a given nucleotide composition all possible oligonucleotide sequences are assembled followed by identification of the correct sequence by the lowest fitness value. Using this concept, sequences of 5- to 12-mer oligodeoxynucleotides were successfully de novo determined. High sequence coverage with fragment ions was essential for obtaining unequivocal sequencing results. Moreover, the collision energy was shown to have an impact on the interpretability of tandem mass spectra by the de novo sequencing algorithm. Experiments revealed that the optimal collision energy should be set to a value just sufficient for complete fragmentation of the precursor ion.     

Generating protein sequence tags by combining cone and conventional collision induced dissociation in a quadrupole time-of-flight mass spectrometer

The goal of proteomics research is to be able to identify and quantify the vast numbers of proteins within an organism or tissue. "Top-down" methods address this goal without the need for proteolytic digestion prior to mass analysis. We report here an approach for top-down protein identification that has been implemented on a commercially available, unmodified Qq-TOF mass spectrometer. Intact protein molecular ions first undergo cone fragmentation in the electrospray inlet. Conventional MS/MS is then performed on a mass selected cone fragment using CID in the Qq interface of the Qq-TOF mass spectrometer to generate a sequence tag through a pseudo-MS3 experiment. Seven proteins varying in molecular weight between 11 and 66 kDa were chosen to demonstrate applicability of method. After the molecular weight of the intact protein was determined, the cone voltage was varied to induce fragmentation. Cone fragment ions were then further dissociated using conventional CID, and the resulting MS/MS spectra were processed and analyzed for sequence tags. Sequence tags were easily identified from a MS/MS spectrum of a cone induced fragment ion both manually and through a de novo sequencing program included in the software associated with the mass spectrometer. Sequence tags were subjected to database searching using the PeptideSearch program of EMBL, and all protein sequence tags gave unambiguous search results. In all cases, sequence tags were found to originate from the n- and/or c-termini of the proteins.