Confirmation and quantification of hemoglobin-based oxygen carriers in equine and human plasma by hyphenated liquid chromatography tandem mass spectrometry

Oxyglobin (OXY) and Hemopure (HMP) are produced from bovine hemoglobin (Hb) and were developed for the treatment of anemia in animal and human patients, respectively. Hemolink (HML) is a blood substitute of human Hb origin under development. The ability of these agents to carry oxygen in circulating blood and their promise to improve oxygen delivery to tissues supports the potential for their abuse in equine and human athletes. To deter athletes from abuse of these agents, a method has been developed for the detection, confirmation and quantification of OXY, HMP, and HML in equine and human plasma. OXY, HMP, and HML were extracted from equine or human plasma by solid-phase extraction using Bond Elut ENV cartridges and were digested by trypsin at 37 degrees C for 3 h. The tryptic digests were analyzed by LC-MS/MS, and tryptic peptides specific for bovine and human Hbs were targeted. OXY and HMP were detected, quantified, and confirmed using the y14 ion and b8 ion of the tryptic peptide from bovine Hb alpha chain residues 69-90, and HML was quantified using the tryptic peptide from human Hb alpha chain residues 63-91. The limit of detection for OXY in equine plasma and HML in human and equine plasma was 50 and 250 microg/mL for HMP in human and equine plasma. The limit of confirmation was 250 microg/mL for OXY in equine plasma, 500 microg/mL for HML in human and equine plasma, and 1000 microg/mL for HMP in human and equine plasma. The linear range for quantification was 50-5000 microg/mL for OXY in equine plasma and for HML in human and equine plasma, and 250-5000 microg/mL for HMP in human and equine plasma. The intraday and interday CV were less than 17% for quantification of OXY in equine plasma with external calibration. OXY was stable for more than 30 days at -20 and -70 degrees C. OXY was detected and quantified in equine plasma up to 24 h following administration of a very low dose of OXY (32.5 g in 2 x 125 mL per horse), and its presence in equine plasma was confirmed up to 12 h postadministration.     

Electrospray tandem mass spectrometry of intact beta-chain hemoglobin variants

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

Doping control analysis of bovine hemoglobin-based oxygen therapeutics in human plasma by LC-electrospray ionization-MS/MS

Since January 2000, hemoglobin-based oxygen carriers, such as Hemopure, belong to the list of prohibited substances of the International Olympic Committee. Hemopure is based on bovine hemoglobin, which is intra- and intermolecularly cross-linked by glutaraldehyde units causing an average molecular weight of approximately 250,000. Bovine and human hemoglobins differ by 15% in amino acid sequence; hence, tryptic digestion of these proteins generates species-common and -unique peptides. Those specific fragments originate from the alpha- and beta-subunits of hemoglobin, such as bovine Hb peptides alpha(69-90) (2367.2 Da) or beta(40-58) (2089.9 Da). By means of LC-MS/MS, peptides of human and bovine hemoglobin can be separated and identified, enabling the determination of compounds based on Hb of bovine origin and thus the administration of oxygen carriers such as Hemopure. Blank plasma samples were spiked with Hemopure or human or bovine hemoglobin, filtered, enzymatically digested, and analyzed on an Agilent 1100 Series HPLC interfaced to an Applied Biosystems API 2000 triple quadrupole mass spectrometer. In plasma aliquots of 50 microL containing 50 microg of Hemopure (1 mg/mL), peptides of bovine hemoglobin were confirmed, and blank plasma samples as well as 68 specimens of high-performance athletes were tested with the developed procedure.     

N-terminal adducts of bovine hemoglobin with glutaraldehyde in a hemoglobin-based oxygen carrier

Hemoglobin-based oxygen carriers (HBOCs) are being developed for the medical field, but because they could increase an athlete's performance, they are misapplied for doping purposes. We previously presented a screening method to detect Oxyglobin (Biopure Corp.) and PolyHeme (Northfield Laboratories Inc.) in serum samples using total acid hydrolysis followed by electrospray mass spectrometry analyses. An alternative mass spectrometric method involving enzymatic hydrolysis is here presented. Digestion of Oxyglobin by endoproteinase Glu-C and LC/MS analyses of the mixture allowed the detection of unique peptidic fragments in comparison with a bovine hemoglobin digest. Tandem mass spectrometry experiments of these peptide ions were performed, and two specific species were actually identified as the N-terminal enzymatic fragment of the beta chain carrying two different modifications. Sequential MS3 experiments using an ion trap mass spectrometer permitted us to locate the chemical modification by the glutaraldehyde on the NH2-terminal group and to propose a structure for the modified peptides. In another set of experiments, screening of these two diagnostic ions into Oxyglobin-spiked serums using precursor ion scan mode in a triple quadrupole instrument allowed the detection of this HBOC with a detection limit of 2 g L(-1).     

LC-MS/MS method for confirmation of recombinant human erythropoietin and darbepoetin alpha in equine plasma

Recombinant human erythropoietin (rhEPO) and darbepoetin alpha (DPO) are protein-based drugs for the treatment of anemia by stimulating red blood cell production. Consequently, they are abused in human and equine sports. To deter their abuse in the horse racing industry, a sensitive and reliable method for confirmation of these agents in equine plasma has been in urgent need. Such a method by LC-MS/MS is described in this paper. The method involved analyte enrichment by immunoaffinity separation using anti-rhEPO antibody linked to magnetic beads, digestion by trypsin, and analysis by LC-MS/MS. Two specific proteotypic peptides, 46VNFYAWK52 and 144VYSNFLR150 from rhEPO and DPO were employed for confirmation of the analytes based on chromatographic retention times and major product ions. The limit of confirmation of this method was 0.2 ng/mL, and the limit of detection was 0.1 ng/mL for rhEPO and DPO in equine plasma. This method was successful in confirming the presence of rhEPO and DPO in plasma samples collected from research horses to which rhEPO or DPO was administered and from racehorses following competition and in noncompetition samples in North America. To our knowledge, this is the first LC-MS method with adequate sensitivity and specificity in providing unequivocal confirmation of rhEPO and DPO in equine plasma samples. This method provides a powerful enforcement tool that was lacking in the fight against the abuse of rhEPO and DPO in the horse racing industry.     

Detecting deamidation products in proteins by electron capture dissociation

A nonenzymatic posttranslational modification of proteins and peptides is the spontaneous deamidation of asparaginyl residues via a succinimide intermediate to form a varying mixture of aspartyl and isoaspartyl residues. The isoaspartyl residue is generally difficult to detect particularly using mass spectrometry because isoaspartic acid is isomeric with aspartic acid so that there is no mass difference. However, electron capture dissociation has demonstrated the ability to differentiate the two isoforms in synthetic peptides using unique diagnostic ions for each form; the cr. + 58 and z(l-r) - 57 fragment ions for the isoAsp form and the Asp side chain loss ((M + nH)(n-1)+. - 60) for the Asp form. Shown here are three examples of isoaspartyl detection in peptides from proteins; a deamidated tryptic peptide of cytochrome c, a tryptic peptide from unfolded and deamidated ribonuclease A, and a tryptic peptide from calmodulin deamidated in its native state. In all cases, the cr. + 58 and z(l-r) - 57 ions allowed the detection and localization of isoaspartyl residues to positions previously occupied by asparaginyl residues. The (M + nH)(n-1)+. - 60 ions were also detected, indicating the presence of aspartyl residues. Observation of these diagnostic ions in peptides from proteins shows that the method is applicable to defining the isomerization state of deamidated proteins.     

Differentiation and identification of recombinant human erythropoietin and darbepoetin Alfa in equine plasma by LC-MS/MS for doping control

Recombinant human erythropoietin (rhEPO) and darbepoetin alfa (DPO) are protein-based drugs for the treatment of anemia in humans by stimulating erythrocyte production. However, these agents are abused in human and equine sports due to their potential to enhance performance. This paper describes the first method for differentiation and identification of rhEPO and DPO in equine plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method comprised analyte extraction and enrichment by immunoaffinity separation with anti-rhEPO antibodies, dual digestion by trypsin and peptide-N-glycosidase F (PNGase F), and analysis by LC-MS/MS. Two unique deglycosylated tryptic peptides, (21)EAENITTGCAEHCSLNENITVPDTK (45) (T 5) from rhEPO and (77)GQALLVNSSQVNETLQLHVDK (97) (T 9) from DPO, were employed for differentiation and identification of rhEPO and DPO via LC retention times and major product ions. The limit of identification was 0.1 ng/mL for DPO and 0.2 ng/mL for rhEPO in equine plasma, and the limit of detection was 0.05 ng/mL for DPO and 0.1 ng/mL for rhEPO. Analyte carryover problem encountered was solved by adding 20% acetonitrile to the solvent of the sample digest to increase solubility of the peptides. This method was successfully applied to identification of DPO in plasma samples collected from a research horse following DPO administration and from racehorses out of competition in North America. Thus, it provides a powerful tool in the fight against blood doping with rhEPO and DPO in the horse racing industry.     

Elution-modified displacement chromatography coupled with electrospray ionization-MS: on-line detection of trace peptides at low-femtomole level in peptide digests

Elution-modified displacement chromatography (EMDC) was employed to achieve peptide separations with high efficiency. On-line ESI-MS and ESI-MS/MS measurements showed enrichment and detection of kemptide, a protein kinase A peptide substrate, at low femtomole levels when it was added as a trace marker component to a tryptic digest of bovine serum proteins or to a human growth hormone peptide digest at concentration ratios between 1:10(5) and 1:10(6). In another EMDC separation, five peptides were detected in a mixture containing 20 fmol of human growth hormone tryptic digest mixed with the bovine serum protein digest. We found that EMDC facilitated rapid detection and sequence analysis of trace peptides at levels of approximately 0.5 fmol/microL in complex peptide mixtures with a wide dynamic concentration range. Accordingly, the detection of kemptide by EMDC was found to be 3-4 orders of magnitude more sensitive than that attained in conventional linear elution chromatography separations performed with the same peptide loads. Kemptide was phosphorylated in vitro and was detected along with its neutral loss product in peptide mixtures at low femtomole levels. EMDC enabled both detection and amino acid sequence determination on trace levels of phosphorylated and other posttranslationally modified peptides, suggesting that the technique may be useful for proteomics applications where detection and analysis of trace level peptides are problematic.     

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

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

Use of performic acid oxidation to expand the mass distribution of tryptic peptides

Significant identification of proteins by mass fingerprinting and partial sequencing of tryptic peptides is central to proteomics. However, peptide masses cluster with distances of approximately 1 Da. Expanding these clusters will give more peptides of unique masses, thereby identifying proteins with a higher significance. The mass clusters can be expanded downward by including more oxygen atoms in the peptides. Classic performic acid oxidation modifies three residues, Cys to CysO(3), Met to MetO(2), and Trp to TrpO(2). In this study, we compare the mass distributions of tryptic peptides computed from the predicted proteomes of Bacillus subtilis, Drosophila melanogaster, Arabidopsis thaliana, and Homo sapiens modified by oxidation, reduction, and reduction followed by carboxymethylation, carboxamidomethylation, or pyridylethylation. Forty to 46% of the eukaryotic tryptic peptides contain Cys, Met, or Trp. Additionally, the importance of mass accuracy of differentially modified tryptic peptides for significant protein identification by database searches was analyzed. The results show that performic acid oxidation gives markedly extended mass distributions at mass accuracies from +/-0.002 to +/-0.25 Da for the eukaryotes. The effect of the expanded mass distribution on significant protein identification was illustrated by searching simulated mass peak lists against the databases containing oxidized and reduced tryptic peptides. The specificity of formic acid oxidation was tested experimentally, and no general adverse effects were detected. Tryptic peptides provided a 100% sequence coverage of oxidized barley grain peroxidase by LC-MS, and the sequence coverages of oxidized and carboxymethylated bovine serum albumin were similar by MALDI-TOF MS analyses.     

Peptide sequence motif analysis of tandem MS data with the SALSA algorithm.

We have developed a pattern recognition algorithm called SALSA (scoring algorithm for spectral analysis) for the detection of specific features in tandem MS (MS-MS) spectra. Application of the SALSA algorithm to the detection of peptide MS-MS ion series enables identification of MS-MS spectra displaying characteristics of specific peptide sequences. SALSA analysis scores MS-MS spectra based on correspondence between theoretical ion series for peptide sequence motifs and actual MS-MS product ion series, regardless of their absolute positions on the m/z axis. Analyses of tryptic digests of bovine serum albumin (BSA) by LC-MS-MS followed by SALSA analysis detected MS-MS spectra for both unmodified and multiple modified forms of several BSA tryptic peptides. SALSA analysis of MS-MS data from mixtures of BSA and human serum albumin (HSA) tryptic digests indicated that ion series searches with BSA peptide sequence motifs identified MS-MS spectra for both BSA and closely related HSA peptides. Optimal discrimination between MS-MS spectra of variant peptide forms is achieved when the SALSA search criteria are optimized to the target peptide. Application of SALSA to LC-MS-MS proteome analysis will facilitate the characterization of modified and sequence variant proteins.     

SALSA: a pattern recognition algorithm to detect electrophile-adducted peptides by automated evaluation of CID spectra in LC-MS-MS analyses

A pattern recognition algorithm called SALSA (scoring algorithm for spectral analysis) has been developed to rapidly screen large numbers of peptide MS-MS spectra for fragmentation characteristics indicative of specific peptide modifications. The algorithm facilitates sensitive and specific detection of modified peptides at low abundance in an enzymatic protein digest. SALSA can simultaneously score multiple user-specified search criteria, including product ions, neutral losses, charged losses, and ion pairs that are diagnostic of specific peptide modifications. Application of SALSA to the detection of peptide adducts of the electrophiles dehydromonocrotaline, benzoquinone, and iodoacetic acid permitted their detection in a complex tryptic peptide digest mixture. SALSA provides superior detection of adducted peptides compared to conventional tandem MS precursor ion or neutral loss scans.     

Mass spectrometry evidence for cisplatin as a protein cross-linking reagent

Cisplatin is a potent anticancer drug, which functions by cross-linking adjacent DNA guanine residues. However within 1 day of injection, 65-98% of the platinum in the blood plasma is protein-bound. It is generally accepted that cisplatin binds to methionine and histidine residues, but what is often underappreciated is that platinum from cisplatin has a 2+ charge and can form up to four bonds. Thus, it has the potential to function as a cross-linker. In this report, the cross-linking ability of cisplatin is demonstrated by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with the use of standard peptides, the 16.8 kDa protein calmodulin (CaM), but was unsuccessful for the 64 kDa protein hemoglobin. The high resolution and mass accuracy of FTICR MS along with the high degree of fragmentation of large peptides afforded by collisionally activated dissociation (CAD) and electron capture dissociation (ECD) are shown to be a valuable means of characterizing cross-linking sites. Cisplatin is different from current cross-linking reagents by targeting new functional groups, thioethers, and imidazoles groups, which provides complementarity with existing cross-linkers. In addition, platinum(II) inherently has two positive charges which enhance the detection of cross-linked products. Higher charge states not only promote the detection of cross-linking products with less purification but result in more comprehensive MS/MS fragmentation and can assist in the assignment of modification sites. Moreover, the unique isotopic pattern of platinum flags cross-linking products and modification sites by mass spectrometry.     

Fragmentation of singly protonated peptides via a combination of infrared and collisional activation

The coupling of matrix-assisted laser desorption/ionization (MALDI) to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides an exceptionally capable platform for peptide analysis, but an important limitation of this approach is the difficulty in obtaining informative tandem mass spectra (MS/MS) of singly protonated peptides. This difficulty is especially pronounced with peptide ions containing basic amino acid residues (for example, tryptic peptides). While such ions can be fragmented in some instrument configurations, most FTICR instruments have comparatively little facility for high-energy fragmentation. Here, a novel MS/MS approach implemented with MALDI-FTICR-MS and specifically intended for enhanced fragmentation of singly protonated peptides is described. The method involves infrared irradiation in concert with the simultaneous application of sustained off-resonance irradiation collision-induced dissociation (SORI-CID). This form of MS/MS, described as a combination of infrared and collisional activation (CIRCA), is shown to provide a greater capacity for dissociation of singly charged model peptide ions as compared to infrared multiphoton dissociation (IRMPD) or SORI-CID alone. Overall, the CIRCA approach is demonstrated to be a feasible technique for accessing useful fragmentation pathways of singly charged peptides, including those harboring basic amino acid residues--a crucial feature in the context of proteomics.     

Combining fluorescence detection and mass spectrometric analysis for comprehensive and quantitative analysis of redox-sensitive cysteines in native membrane proteins

Monobromobimane (MBB) is a lipophilic reagent that selectively modifies free cysteine residues in proteins. Because of its lipophilic character, MBB is capable of labeling cysteine residues in membrane proteins under native conditions. Reaction of MBB with the sulfhydryl groups of free cysteines leads to formation of highly fluorescent derivatives. Here we describe a procedure for the detection and relative quantitation of MBB-labeled cysteines using fluorescence and mass spectrometric analyses, which allow determination of free cysteine content and unambiguous identification of MBB-modified cysteine residues. We have applied this approach to the analysis of the free and redox-sensitive cysteine residues of a large membrane protein, the sarcoplasmic reticulum Ca2+ release channel with a molecular mass of 2.2 million Da. Labeling was performed under physiologic conditions where the channel complex is in its native environment and is functionally active. The purified MBB-labeled channel complex was enzymatically digested, and the resulting peptides were separated by reversed-phase high-performance chromatography. MBB-labeled peptides were detected by fluorescence and identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Under MALDI conditions, partial photolytic fragmentation of the MBB-peptide bound occurred, thus allowing convenient screening for the MBB-modified peptides in the MS spectrum by detection of the specific mass increment of 190.07 Da for MBB-modified cysteine residues. Modification of the peptides was further confirmed by tandem mass spectrometric analysis, utilizing sequencing information and the presence of the specific immonium ion for the MBB-modified cysteine residues at m/z 266.6. Quantitative information was obtained by comparison of both fluorescence and MS signal intensities of MBB-modified peptides. Combination of fluorescence with MS detection and analysis of MBB-labeled peptides supported by a customized software program provides a convenient method for identifying and quantifying redox-sensitive cysteines in membrane proteins of native biological systems. Identification of one redox-sensitive cysteine (2327) in the native membrane-bound sarcoplasmic reticulum Ca2+ release channel is described.     

Ultrahigh-throughput proteomics using fast RPLC separations with ESI-MS/MS

We describe approaches for proteomics analysis using electrospray ionization-tandem mass spectrometry coupled with fast reversed-phase liquid chromatography (RPLC) separations. The RPLC separations used 50-microm-i.d. fused-silica capillaries packed with submicrometer-sized C18-bonded porous silica particles and achieved peak capacities of 130-420 for analytes from proteome tryptic digests. When these separations were combined with linear ion trap tandem mass spectrometry measurements, approximately 1000 proteins could be identified in 50 min from approximately 4000 identified tryptic peptides; approximately 550 proteins in 20 min from approximately 1800 peptides; and approximately 250 proteins in 8 min from approximately 700 peptides for a S. oneidensis tryptic digest. The dynamic range for protein identification with the fast separations was determined to be approximately 3-4 orders of magnitude of relative protein abundance on the basis of known proteins in human blood plasma analyses. We found that 55% of the MS/MS spectra acquired during the entire analysis (and up to 100% of the MS/MS spectra acquired from the most data-rich zone) provided sufficient quality for identifying peptides. The results confirm that such analyses using very fast (minutes) RPLC separations based on columns packed with microsized porous particles are primarily limited by the MS/MS analysis speed.     

Statistical characterization of the charge state and residue dependence of low-energy CID peptide dissociation patterns

Data mining was performed on 28 330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chemical interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx-Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissociation patterns for singly charged Arg- and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides, a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from singly charged peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s) in multiply protonated peptides. Enhanced cleavages C-terminal to branched aliphatic residues (Ile, Val, Leu) are observed in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages N-terminal to these residues are observed in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns observed expand our knowledge of peptide gas-phase fragmentation behaviors and may be useful in algorithm development that employs improved models to predict fragment ion intensities.     

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

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

Phosphorylation site identification via ion trap tandem mass spectrometry of whole protein and peptide ions: bovine alpha-crystallin A chain

Tandem mass spectrometry was applied both to ions of a tryptic fragment and intact protein of bovine alpha-crystallin A chain to localize the single site of phosphorylation. The [M + 19H](19+) to [M + 11H](11+) charge states of both phosphorylated and unphosphorylated bovine alpha-crystallin A chain whole protein ions were subjected to collisional activation in a quadrupole ion trap. Ion parking was used to increase the number of parent ions over that yielded by electrospray. Ion-ion proton-transfer reactions were used to reduce the product ion charge states largely to +1 to simplify spectral interpretation. In agreement with previous studies on whole protein ion fragmentation, both protein forms showed backbone cleavages C-terminal to aspartic acid residues at lower charge states. The phosphorylated protein showed competitive fragmentation between backbone cleavage and the neutral loss of phosphoric acid. Analysis of which backbone cleavage products did or did not contain the phosphate was used to localize the site of phosphorylation to one of two possible serine residues. A tryptic digest of the bovine alpha-crystallin A chain yielded a phosphopeptide containing one missed cleavage site. The peptide provided information complementary to that obtained from the intact protein and localized the modified serine to residue 122. Fragmentation of the triply charged phosphopeptide yielded five possible serine phosphorylation sites. Fragmentation of the doubly charged phosphopeptide, formed by ion/ion proton-transfer reactions, positively identified the phosphorylation site as serine-122.     

Investigation of neutral loss during collision-induced dissociation of peptide ions

MS/MS fragmentation of peptides is dominated by overlapping b and y ion series. However, alternative fragmentation possibilities exist, including neutral loss. A database was generated containing 8400 MS/MS spectra of tryptic peptides assigned with high probability to an amino acid sequence (true positives) and a set of certified false (true negative) assignments for analysis of the amino terminus. A similar database was created for analysis of neutral loss at the carboxy termini using a data set of chymotryptic peptides. The analysis demonstrated that the presence of an internal basic residue, limiting proton mobility, has a profound effect on neutral loss. Peptides with fully mobile protons demonstrated minimal neutral loss, with the exception of amide bonds with proline on the carboxy terminal side, which created an intense neutral loss peak. In contrast, peptides with partial proton mobility contained many amino acids on either side of the amide bond associated with a strong neutral loss peak. Most notable among these was proline on the carboxy terminal side of an amide bond and aspartic acid on the amino terminal side of a bond. All results were found to be consistent for doubly and triply charged peptides and after adjustment for pairings across the amide bonds with particularly labile residues. The carboxy terminal of chymotryptic peptides also demonstrated significant neutral loss events associated with numerous amino acid residues. Clarification of the rules that govern neutral loss, when incorporated into analysis software, will improve our ability to correctly assign spectra to peptide sequences.