Improved detection of intact tyrosine sulfate-containing peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in linear negative ion mode

Sulfation of tyrosine residues is a common post-translational modification, but detecting and quantitating this modification poses challenges due to lability of the sulfate group. The goal of our studies was to determine how best to detect and to assess the stoichiometry of this modification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Sulfated and nonsulfated forms of peptides—hirudin(55–65), caerulein, and cholecystokinin octapeptide and phosphorylated and nonphosphorylated pp60-c-src (521–533)—were analyzed using several matrices: sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DBA), and cyano-4-hydroxycinnamic acid (CHCA). Intact sulfated peptides were difficult to detect using positive ion mode; peptides were observed as desulfated ions. Phosphorylated peptide was stable and was detected in positive and negative ion modes. Detection of sulfated peptides improved with: (1) Analysis in negative ion mode, (2) Decreased laser power, (3) Matrix selection: DBA ≥ SA > CHCA. In negative ion mode, desorption/ionization of sulfated peptide was equivalent or more efficient than nonsulfated peptide, depending on conditions of analysis. Examination of a tryptic digest of α₂-antiplasmin detected the single site of sulfation in negative ion mode but not in positive ion mode. We conclude that improved detection of sulfated peptides can be achieved in negative ion mode. Dual analysis in positive and negative ion modes serves as a potential means of identifying peptides with labile modifications such as sulfation and distinguishing them from phosphorylation.     

Identification of phosphopeptides by MALDI Q-TOF MS in positive and negative ion modes after methyl esterification

We have developed an efficient, sensitive, and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by CID of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification before mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of beta-casein and alpha-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides.     

Comparison of the electron capture dissociation fragmentation behavior of doubly and triply protonated peptides from trypsin, Glu-C, and chymotrypsin digestion

In bottom-up proteomics, proteolytically derived peptides from proteins of interest are analyzed to provide sequence information for protein identification and characterization. Electron capture dissociation (ECD), which provides more random cleavages compared to "slow heating" techniques such as collisional activation, can result in greater sequence coverage for peptides and proteins. Most bottom-up proteomics approaches rely on tryptic doubly protonated peptides for generating sequence information. However, the effectiveness, in terms of peptide sequence coverage, of tryptic doubly protonated peptides in ECD remains to be characterized. Herein, we examine the ECD fragmentation behavior of 64 doubly- and 64 triply protonated peptides (i.e., a total of 128 peptide ions) from trypsin, Glu-C, and chymotrypsin digestion in a Fourier transform ion cyclotron resonance mass spectrometer. Our findings indicate that when triply protonated peptides are fragmented in ECD, independent of which proteolytic enzyme was used for protein digestion, more c- and z-type product ions are observed, and the number of complementary fragment pairs increases dramatically (44%). In addition, triply protonated peptides provide an increase (26%) in peptide sequence coverage. ECD of tryptic peptides, in both charge states, resulted in higher sequence coverage compared to chymotryptic and Glu-C digest peptides. The peptide sequence coverage we obtained in ECD of tryptic doubly protonated peptides (64%) is very similar to that reported for electron transfer dissociation of the same peptide type (63%).     

Degradation and debittering of a tryptic digest from beta-casein by aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2.

The mode of action of purified aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 on a complex peptide mixture of a tryptic digest from bovine beta-casein was analyzed. The oligopeptides produced in the tryptic digest before and after aminopeptidase N treatment were identified by analysis of the N- and C-terminal amino acid sequences and amino acid compositions of the isolated peptides and by on-line liquid chromatography-mass spectrometry. Incubation of purified peptides with aminopeptidase N resulted in complete hydrolysis of many peptides, while others were only partially hydrolyzed or not hydrolyzed. The tryptic digest of beta-casein exhibits a strong bitter taste, which corresponds to the strong hydrophobicity of several peptides in the tryptic digest of beta-casein. The degradation of the "bitter" tryptic digest by aminopeptidase N resulted in a decrease of hydrophobic peptides and a drastic decrease of bitterness of the reaction mixture.     

Characteristic mass spectral fragments of the organophosphorus agent FP-biotin and FP-biotinylated peptides from trypsin and bovine albumin (Tyr410)

A mass spectrometry-based method was developed for selective detection of FP-biotinylated peptides in complex mixtures. Mixtures of peptides, at the low-picomole level, were analyzed by liquid chromatography and positive ion, nanospray, triple quadrupole, linear ion trap mass spectrometry. Peptides were fragmented by collision-activated dissociation in the mass spectrometer. The free FP-biotin and peptides containing FP-biotinylated serine or FP-biotinylated tyrosine yielded characteristic fragment ions at 227, 312, and 329 m/z. FP-biotinylated serine yielded an additional characteristic fragment ion at 591 m/z. Chromatographic peaks containing FP-biotinylated peptides were indicated by these diagnostic ions. Data illustrating the selectivity of the approach are presented for tryptic digests of FP-biotinylated trypsin and FP-biotinylated serum albumin. A 16-residue peptide from bovine trypsin was biotinylated on the active site serine. A 3-residue peptide from bovine albumin, YTR, was biotinylated on Tyr410. This latter result confirms that the organophosphorus binding site of albumin is a tyrosine. This method can be used to search for new biomarkers of organophosphorus agent exposure.     

High sequence coverage by in-capillary proteolysis of native proteins and simultaneous analysis of the resulting peptides by nanoelectrospray ionization-mass spectrometry and tandem mass spectrometry

Losses of proteolytic peptides during extraction and/or purification procedures succeeding in-gel or in-solution digests of proteins frequently occur in the course of protein identification investigations. In order to overcome this disadvantage, the method of in-capillary digest was developed: native proteins were incubated in the presence of endoproteases in the electrospray capillary and the resulting peptides were analyzed by nanoelectrospray-mass spectrometry during the ongoing proteolysis. In-capillary digest of apomyglobin by use of trypsin in a molar ratio of 25:1 yielded complete degradation already after 15 min. The sequence coverage based on formation of molecular ions was 100% and peptide ions could be fragmented by collision-induced dissociation and sequenced. When myoglobin was incubated in the electrospray capillary with trypsin in a molar ratio of 500:1, a clear shift from molecular ions and miscleaved peptide ions to the expected final tryptic peptide ions was observed over a 2 h period. The peptide spectra obtained from tryptic in-capillary proteolysis of bovine serum albumin and apotransferrin, respectively, gave rise to sequence coverages of more than 40% for both proteins. The data obtained from the peptide maps as well as from collision-induced dissociation (CID) of selected peptides were more than sufficient for protein identification by database searches. An elephant milk protein preparation was used to demonstrate the application of in-capillary proteolysis on protein mixtures. Tryptic digest, simultaneous analysis of the proteolytic peptides by use of CID, and subsequent sequencing allowed the identification of lactoferrin, alphas1-casein, beta-casein, delta-casein, and kappa-casein by homology search.     

Mapping the ankyrin-binding site of the human erythrocyte anion exchanger

This report describes initial efforts to map the ankyrin-binding site of the cytoplasmic domain of the human erythrocyte anion exchanger. The conclusions are that this site is likely to involve a fairly extended sequence in the midregion of the cytoplasmic domain and requires interactions that are not provided by isolated peptides. The region of the sequence involving residues 174-186 is likely to participate in the ankyrin-binding site based on several experiments. Limited tryptic cleavage in the midregion of the cytoplasmic domain (residues 174 and/or 181) nearly abolished the ability of the cytoplasmic domain to inhibit binding of ankyrin to the anion exchanger. Ankyrin protected the cytoplasmic domain from tryptic digestion. Finally, peptide-specific antibodies against the sequence encompassing the site(s) of tryptic cleavage (residues 174-186) blocked binding of ankyrin to the anion exchanger. However, the sequence comprising the tryptic site is not sufficient for high affinity binding of ankyrin. A 39-amino acid peptide (residues 161-200) that includes the tryptic cleavage site(s) was inactive in inhibiting binding of ankyrin to the anion exchanger. Further evidence for a complex ankyrin-binding site is that peptide-specific antibodies against two different, noncontiguous regions (residues 118-162 and 174-186) both inhibited binding of ankyrin to the anion exchanger and were only 10-20% as effective as antibody against the entire cytoplasmic domain. Finally, the ankyrin-binding site of the anion exchanger did not renature following sodium dodecyl sulfate electrophoresis and transfer to nitrocellulose paper even though spectrin did recover ability to bind ankyrin under the same conditions. Thus, the ankyrin-binding site is not defined by a short continuous sequence. A simple consensus sequence for ankyrin-binding regions in other proteins is not likely.     

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 disulfide-containing peptides by performic acid oxidation and mass spectrometry

In addition to reducing the analysis time, the direct examination of proteolytic digests by fast atom bombardment mass spectrometry (FABMS) greatly extends the information that is available from peptide mapping experiments. Mass spectral data are particularly useful for identifying post-translationally modified peptides. For example, the molecular weight of a disulfide-containing peptide may be used to locate the disulfide bond in the protein from which the peptide was derived. This paper describes a new procedure, which is useful for identifying disulfide-bonded peptides. Peptides are treated with performic acid to modify certain residues and thereby cause a characteristic change in the peptide molecular weight. This change in molecular weight is determined by FABMS and used to help identify peptides. Results for a series of small peptides demonstrate that Cys, Met, and Trp are the only residues that undergo a change in molecular weight under the conditions used here. Furthermore, these changes in molecular weight are diagnostic for each of the residues. Cysteinyl-containing peptides are of particular interest, because their identification is essential for locating disulfide bonds. The molecular weight of a peptide increases by 48 mu for each cysteinyl residue present. This approach is used to identify peptides that contain both cysteinyl and cystinyl residues in the peptic digest of bovine insulin. The method is extended to the analysis of a tryptic digest of cyanogen bromide-treated ribonuclease A. A computer-assisted analysis procedure is used to demonstrate the specificity with which peptide molecular weight is related to specific segments of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos

Over the past decade peptide sequencing by collision induced dissociation (CID) has become the method of choice in mass spectrometry-based proteomics. The development of alternative fragmentation techniques such as electron transfer dissociation (ETD) has extended the possibilities within tandem mass spectrometry. Recent advances in instrumentation allow peptide fragment ions to be detected with high speed and sensitivity (e.g., in a 2D or 3D ion trap) or at high resolution and high mass accuracy (e.g., an Orbitrap or a ToF). Here, we describe a comprehensive experimental comparison of using ETD, ion-trap CID, and beam type CID (HCD) in combination with either linear ion trap or Orbitrap readout for the large-scale analysis of tryptic peptides. We investigate which combination of fragmentation technique and mass analyzer provides the best performance for the analysis of distinct peptide populations such as N-acetylated, phosphorylated, and tryptic peptides with up to two missed cleavages. We found that HCD provides more peptide identifications than CID and ETD for doubly charged peptides. In terms of Mascot score, ETD FT outperforms the other techniques for peptides with charge states higher than 2. Our data shows that there is a trade-off between spectral quality and speed when using the Orbitrap for fragment ion detection. We conclude that a decision-tree regulated combination of higher-energy collisional dissociation (HCD) and ETD can improve the average Mascot score.     

Identification of tyrosine sulfation in extracellular leucine-rich repeat proteins using mass spectrometry

Multiple and variable tyrosine sulfation in extracellular class II leucine-rich repeat proteins/proteoglycans were characterized by mass spectrometry. The sulfogroup on tyrosine is labile and is released from peptides under normal mass spectrometric conditions. Thus, special approaches must be considered in order to identify this modification. By using a combination of mass spectrometry studies operating in negative and positive ion mode, tyrosine sulfation could be identified. In positive mode, the peptides normally appeared non-sulfated, whereas in negative mode a mixture of sulfated and non-sulfated species was observed. A combination of peptides released by different proteinases was used to obtain details on the locations of sulfate groups. Multiple tyrosine sulfates were observed in the N-terminal region of fibromodulin (up to 9 sites), osteoadherin (up to 6 sites), and lumican (2 sites). Osteoadherin contains two additional sulfated tyrosine residues close to its C terminus. We also identified an error in the published sequence of bovine fibromodulin, resulting in the replacement of Thr37 by Tyr37-Gly38, thus increasing its homology with its human counterpart.     

Extended Range Proteomic Analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of beta-casein and epidermal growth factor receptor (EGFR)

We have developed a new and sensitive LC-MS platform, Extended Range Proteomic Analysis (ERPA), which is able to achieve very high sequence coverage and comprehensive characterization of post-translational modifications in complex proteins. This new platform provides advantages of both the top-down and bottom-up proteomic approaches by combining (i) digestion of the protein with an enzyme, such as Lys-C, which cuts less frequently than trypsin, leading to on average a higher molecular weight peptide size, (ii) high-performance LC separation of the resulting fragments, (iii) a new data acquisition strategy using the LTQ-FTMS, a hybrid mass spectrometer that couples a linear ion trap with a Fourier transform ion cyclotron resonance (FTICR) cell, for analysis of peptides in the range of 0.5 to 10 kDa, and (iv) new data analysis methods for assigning large peptide structures and determining the site of attachment of post-translational modifications as well as structural features from the accurate precursor mass together with MS(2) and MS(3) fragmentations. The LC retention of the Lys-C fragments is increased, relative to a tryptic digest, due to the generally greater hydrophobicity of the larger peptides, a result that is particularly important for peptides containing hydrophilic modifications such as glycosylation and phosphorylation. Furthermore, additional positively charged arginine and lysine residues in the Lys-C fragments enhance the sensitivity of the post-translationally modified phospho- and glycopeptides by at least 10-fold relative to tryptic fragments. In typical operation, the FTICR cell provides a survey scan with the high mass resolution (> 100 000) and accurate mass (<2 ppm) to characterize the higher charge-state precursor ions of the larger peptides. In parallel, the linear ion trap provides MS(2) and MS(3) fragmentation spectra, with a scan speed sufficiently fast for on-line LC-MS. Together, these data provide multiple means to determine or enhance the confidence of assignment of large or complicated peptide. Using ERPA, we demonstrate >95% sequence coverage in the analysis of two heavily phosphorylated and glycosylated proteins, beta-casein at the 50 fmole level and the epidermal growth factor receptor (EGFR) at the 1 pmole level. In summary, the combination of digestion strategy, high-performance separation, and the hybrid LTQ-FTMS instrument enables comprehensive characterization of large proteins, including posttranslational modifications.     

Purification of tryptic peptides for mass spectrometry using polyvinylidene fluoride membrane.

A simple procedure for the purification of tryptic peptides, prior to mass spectrometric analysis, using polyvinylidene fluoride membrane (PVDF) is described. The sensitivity of mass spectrometric analysis is such that minor impurities in tryptic peptide digests suppress the signal obtained. However, we obtained useful signal, from a sample that did not yield any spectra earlier, by purifying the sample using PVDF membrane. For this, the tryptic peptide digest was first spotted on the membrane which was then air-dried and washed. Further, the membrane was extracted with trifluoroacetic acid (TFA) and acetonitrile and subjected to mass spectrometric analysis. This procedure enabled us to identify a cross-reactive D1 antigen on the neutrophil surface that bound antibodies that targeted 60 kD Ro autoantigen in systemic lupus erythematosus, an autoimmune disorder.     

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.     

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.     

Reaction of phosphorylated and O-glycosylated peptides by chemically targeted identification at ambient temperature.

Conditions for carrying out chemically targeted identification of peptides containing phosphorylated or glycosylated serine residues have been investigated. Ba(OH)2 was used at ambient temperature to catalyze the beta-elimination reaction at 25 degrees C. Nucleophilic addition of 2-aminoethanethiol was performed in both parallel and tandem experiments. The method was demonstrated by the reaction of beta-casein tryptic digest phosphopeptides and an O-glycosylated peptide. Contrary to an earlier report by others, the glycopeptide was found to react with essentially the same kinetics as phosphopeptides. Conversion of four phosphoserines in residues 15, 17, 18, and 19 from bovine beta-casein N-terminal tryptic phosphopeptides were followed by monitoring the time course of the addition reaction. The chemistry proceeded rapidly at room temperature with a half-reaction time of 15 min. No side-reaction products were observed; however, care was taken to minimize all counter ions that either precipitate barium or neutralize the base. Digestion of the converted peptides with lysine endopeptidase identified all five phosphoserines in the beta-casein tryptic digest. Alternatively, preincubation with base followed by nucleophilic addition of the thiol was found to work satisfactorily. The use of the water-soluble hydrochloride of 2-aminoethanethiol allowed beta-elimination, nucleophilic addition, and desalting to be carried out on a micro C18 reverse phase pipette tip.     

Characterization of the catalase-peroxidase KatG from Burkholderia pseudomallei by mass spectrometry

The electron density maps of the catalase-peroxidase from Burkholderia pseudomallei (BpKatG) presented two unusual covalent modifications. A covalent structure linked the active site Trp111 with Tyr238 and Tyr238 with Met264, and the heme was modified, likely by a perhydroxy group added to the vinyl group on ring I. Mass spectrometry analysis of tryptic digests of BpKatG revealed a cluster of ions at m/z 6585, consistent with the fusion of three peptides through Trp111, Tyr238, and Met264, and a cluster at m/z approximately 4525, consistent with the fusion of two peptides linked through Trp111 and Tyr238. MS/MS analysis of the major ions at m/z 4524 and 4540 confirmed the expected sequence and suggested that the multiple ions in the cluster were the result of multiple oxidation events and transfer of CH3-S to the tyrosine. Neither cluster of ions at m/z 4525 or 6585 was present in the spectrum of a tryptic digest of the W111F variant of BpKatG. The spectrum of the tryptic digest of native BpKatG also contained a major ion for a peptide in which Met264 had been converted to homoserine, consistent with the covalent bond between Tyr238 and Met264 being susceptible to hydrolysis, including the loss of the CH3-S from the methionine. Analysis of the tryptic digests of hydroperoxidase I (KatG) from Escherichia coli provided direct evidence for the covalent linkage between Trp105 and Tyr226 and indirect evidence for a covalent linkage between Tyr226 and Met252. Tryptic peptide analysis and N-terminal sequencing revealed that the N-terminal residue of BpKatG is Ser22.     

The Essential Role of Mass Spectrometry in Characterizing Protein Structure: Mapping Posttranslational Modifications

Over the last few years we have developed mass spectrometry-based approaches for selective identification of a variety of posttranslational modifications, and for sequencing the modified peptides. These methods do not involve radiolabeling or derivatization. Instead, modification-specific fragment ions are produced by collision-induced dissociation (CID) during analysis of peptides by ESMS. The formation and detection of these marker ions on-the-fly during the LC-ESMS analysis of a protein digest is a powerful technique for identifying posttranslationally modified peptides. Using the marker ion strategy in an orthogonal fashion, a precursor ion scan can detect peptides which give rise to a diagnostic fragment ion, even in an unfractionated protein digest. Once the modified peptide has been located, the appropriate precursor ion can be sequenced by tandem MS. The utility and interplay of this approach to mapping PTM is illustrated with examples that involve protein glycosylation and phosphorylation.     

Characterization of a new fetal hemoglobin variant, Hb F Izumi A gamma 6Glu replaced by Gly, by molecular secondary ion mass spectrometry

Molecular secondary ion mass spectrometry has characterized the structure of a new fetal hemoglobin variant, Hb F Izumi, without separation of peptides or amino acid analysis. First, the mass spectrum of a tryptic digest of the abnormal gamma globin revealed a decreased by 72 mass units in the molecular mass of peptide T-1,2, indicating the presence of a Glu leads to Gly substitution. Next, the analysis of the digest produced by the addition of staphylococcal protease, which specifically cleaves glutamyl peptide bonds, determined the site of substitution at 6th glutamic acid residue in peptide T-1,2 which contains two glutamic acid residues. Since this mass spectrometric approach provides digitalized data on peptide analysis, we call it 'digit printing'. The high sensitivity of this technique is especially promising for the analysis of molecular abnormality in various genetic disorders.     

Peptide end sequencing by orthogonal MALDI tandem mass spectrometry

Highly sensitive peptide fragmentation and identification in sequence databases is a cornerstone of proteomics. Previously, a two-layered strategy consisting of MALDI peptide mass fingerprinting followed by electrospray tandem mass spectrometry of the unidentified proteins has been successfully employed. Here, we describe a high-sensitivity/high-throughput system based on orthogonal MALDI tandem mass spectrometry (o-MALDI) and the automated recognition of fragments corresponding to the N- and C-terminal amino acid residues. Robotic deposition of samples onto hydrophobic anchor substrates is employed, and peptide spectra are acquired automatically. The pulsing feature of the QSTAR o-MALDI mass spectrometer enhances the low mass region of the spectra by approximately 1 order of magnitude. Software has been developed to automatically recognize characteristic features in the low mass region (such as the y1 ion of tryptic peptides), maintaining high mass accuracy even with very low count events. Typically, the sum of the N-terminal two ions (b2 ion), the third N-terminal ion (b3 ion), and the two C-terminal fragments of the peptide (y1 and y2) can be determined. Given mass accuracy in the low ppm range, peptide end sequencing on one or two tryptic peptides is sufficient to uniquely identify a protein from gel samples in the low silver-stained range.