Asp-N Digestion Research Articles

PE/PPE Proteome and ESX-5 Substrate Spectrum in Mycobacterium marinum.

PE/PPE proteins secreted by the ESX-5 type VII secretion system constitute a major protein repertoire in pathogenic mycobacteria and are essential for bacterial survival, pathogenicity, and host-pathogen interaction; however, little is known about their expression and secretion. The scarcity of arginine and lysine residues in PE/PPE protein sequences and the high homology of their N-terminal domains limit protein identification using classical trypsin-based proteomic methods. This study used endoproteinase AspN and trypsin to characterize the proteome of Mycobacterium marinum. Twenty-seven PE/PPE proteins were uniquely identified in AspN digests, especially PE_PGRS proteins. These treatments allowed the identification of approximately 50% of the PE/PPE pool encoded in the genome. Moreover, EspG5 pulldown assays retrieved 44 ESX-5-associated PPE proteins, covering 85% of the PPE pool in the identified proteome. The identification of PE/PE_PGRS proteins in the EspG5 interactome suggested the presence of PE-PPE pairs. The correlation analysis between protein abundance and phylogenetic relationships found potential PE/PPE pairs, indicating the presence of multiple PE/PE_PGRS partners in one PPE. We validated that EspG5 interacted with PPE31 and PPE32 and mapped critical residues for complex formation. The modified proteomic platform increases the coverage of PE/PPE proteins and elucidates the expression and localization of these proteins.

One-Pot Quantitative Top- and Middle-Down Analysis of GluC-Digested Histone H4.

Histone post-translational modifications (PTMs) have been intensively investigated due to their essential function in eukaryotic genome regulation. Histone modifications have been effectively studied using modified bottom-up proteomics approaches; however, the methods often do not capture single-molecule combinations of PTMs (proteoforms) that mediate known and expected biochemical mechanisms. Both middle-down mass spectrometry (MS) and top-down MS quantitation of H4 proteoforms present viable access to this important information. Histone H4 middle-down has previously avoided GluC digestion due to complex digestion products and interferences; however, the common AspN digestion cleaves at amino acid 23, disconnecting K31ac from other PTMs. Here, we demonstrate the effective use of GluC-based middle-down quantitation and compare it to top-down-based quantitation of proteoforms. Despite potential interferences in the m/z space, the proteoforms arising from all three GluC products (E52, E53, and E63) and intact H4 are chromatographically resolved and successfully analyzed in a single LC-MS analysis. Quantitative results and associated analytical metrics are compared between the different analytes of a single sample digested to different extents to reveal general concordance as well as the relative biases and complementarity of each approach. There is moderate proteoform discordance between digestion products (e.g., E52 and E53); however, each digestion product exhibits high concordance, regardless of digestion time. Under the conditions used, the GluC products are better chromatographically resolved yet show greater variance than the top-down quantitation that are more extensively sampled for MS2. GluC-based middle-down of H4 is thus viable. Both top-down and middle-down approaches have comparable quantitation capacity and are complementary.

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding

Mutations in hemoglobin can cause a wide range of phenotypic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin α chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) → Leu mutation in α1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (∼16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the α H58L replacement. Mutant α subunits containing Leu-58(E7) autoxidize ∼8 times and lose hemin ∼200 times more rapidly than native α subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the α H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O2, promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant α subunit has an ∼80,000-fold higher affinity for CO than O2, causing it to rapidly take up and retain carbon monoxide, which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.

Comparative Proteomics of Mouse Tears and Saliva: Evidence from Large Protein Families for Functional Adaptation.

We produced a tear proteome of the genome mouse, C57BL/6, that contained 139 different protein identifications: 110 from a two-dimensional (2D) gel with subsequent trypsin digestion, 19 from a one-dimensional (1D) gel with subsequent trypsin digestion and ten from a 1D gel with subsequent Asp-N digestion. We compared this tear proteome with a C57BL/6 mouse saliva proteome produced previously. Sixteen of the 139 tear proteins are shared between the two proteomes, including six proteins that combat microbial growth. Among the 123 other tear proteins, were members of four large protein families that have no counterparts in humans: Androgen-binding proteins (ABPs) with different members expressed in the two proteomes, Exocrine secreted peptides (ESPs) expressed exclusively in the tear proteome, major urinary proteins (MUPs) expressed in one or both proteomes and the mouse-specific Kallikreins (subfamily b KLKs) expressed exclusively in the saliva proteome. All four families have members with suggested roles in mouse communication, which may influence some aspect of reproductive behavior. We discuss this in the context of functional adaptation involving tear and saliva proteins in the secretions of mouse lacrimal and salivary glands, respectively.

Ion-pair reversed-phase high performance liquid chromatography method for the quantification of isoaspartic acid in a monoclonal antibody

Isomerization of aspartic acid residues is one of the major causes of chemical degradation during the shelf life of biological pharmaceuticals. Monoclonal antibody biopharmaceuticals are typically stored at mildly acidic pH conditions, which can lead to the isomerization reaction. The mechanism of this non-enzymatic chemical reaction has been studied in great detail. However, the identification and quantification of the isomerization sites in a given protein still remains a challenge. We developed an ion-pair reversed-phase HPLC method for the separation of an intact monoclonal antibody variant containing a single isoaspartic acid residue from its native counterpart. We identified and characterized the isomerization site using ion-pair reversed-phase HPLC mass spectrometry methods of the reduced and alkylated antibody and the enzymatically cleaved antibody. Lys-C followed by Asp-N digestion of the antibody was used for the identification of the isomerization site. Electron transfer dissociation (ETD) mass spectrometry was used to confirm the isomerization site at a DY motif at an aspartic acid residue in the CDR-H3 region of the antibody. Tyrosine at the C-terminus of an aspartic acid residue is typically not regarded as a hot spot for isomerization. Our findings suggest that it is not possible to predict isomerization sites in proteins with confidence and all aspartic acid residues located in the CDR regions of antibodies must be considered as potential isomerization site due to the solvent exposure or the flexibility of these regions of the molecule. Additionally, the effect of the pH on the isomerization rate was evaluated using the ion-pair reversed-phase HPLC method, showing that at a lower pH the isomerization rate is faster. Storage at 25°C for 6 months resulted in an increase of the amount of isoaspartic acid to 6.6% at pH 5.4, 6.0% at pH 5.8, and 5.6% at pH 6.2.

Selective isolation of N-blocked peptide by combining AspN digestion, transamination, and tosylhydrazine glass treatment

Many eukaryotic proteins are blocked at the α-amino group of their N-terminal with various modifications, thereby making it difficult to determine their N-terminal sequence by protein sequencer. We propose a novel method for selectively isolating the blocked N-terminal peptide from the peptide mixture generated by endoproteinase AspN digestion of N-blocked protein. This method is based on removal of all peptides other than the N-terminal one (non-N-terminal peptides) through their carbonyl group introduced by a chemical transamination reaction. The transamination reaction converts the free α-amino group of the non-N-terminal peptides to a carbonyl group, whereas the blocked N-terminal peptide, which lacks only the free α-amino group, remains unchanged. Silica functionalized with the tosylhydrazino group effectively captures non-N-terminal peptides through their carbonyl group; thus, the blocked N-terminal peptide is selectively recovered in the supernatant. This method was applied to several model proteins, and their N-terminal peptides were successfully isolated and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, the method was extended to N-terminal analysis of N-free protein by artificially blocking the free α-amino group of its N-terminal with N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide reagent.

Analysis of Isoaspartic Acid by Selective Proteolysis with Asp-N and Electron Transfer Dissociation Mass Spectrometry

A ubiquitous yet underappreciated protein post-translational modification, isoaspartic acid (isoAsp, isoD, or beta-Asp), generated via the deamidation of asparagine or isomerization of aspartic acid in proteins, plays a diverse and crucial role in aging, as well as autoimmune, cancer, neurodegeneration, and other diseases. In addition, formation of isoAsp is a major concern in protein pharmaceuticals, as it may lead to aggregation or activity loss. The scope and significance of isoAsp have, up to now, not been fully explored, as an unbiased screening of isoAsp at low abundance remains challenging. This difficulty is due to the subtle difference in the physicochemical properties between isoAsp and Asp, e.g., identical mass. In contrast, endoprotease Asp-N (EC 3.4.24.33) selectively cleaves aspartyl peptides but not the isoaspartyl counterparts. As a consequence, isoaspartyl peptides can be differentiated from those containing Asp and also enriched by Asp-N digestion. Subsequently, the existence and site of isoaspartate can be confirmed by electron transfer dissociation (ETD) mass spectrometry. As little as 0.5% of isoAsp was detected in synthetic beta-amyloid and cytochrome c peptides, even though both were initially assumed to be free of isoAsp. Taken together, our approach should expedite the unbiased discovery of isoAsp.

Dipeptidyl peptidase 9 (DPP9) from bovine testes: Identification and characterization as the short form by mass spectrometry

The dipeptidyl peptidases (DPP) 8 and 9 belong to the DPP4 activity and/or structure homologues (DASH). Recently, a DPP9-like protein was purified from bovine testes. The aim of the present study was to prove its identity and to investigate the characteristics of this natural enzyme. We report the identification and N-terminal sequence analysis by MALDI-TOF/TOF MS, of the purified bovine enzyme as DPP9. The tryptic peptides after in-gel digestion covered 41% and 38% of the short and full-length variants of bovine DPP9, respectively. Using Asp-N digestion combined with a very recently described mass spectrometric method using DITC glass beads, the N-terminal peptide (XTGALTSERG) was isolated. It corresponds to the N-terminus of the short form of bovine DPP9. There was no evidence for glycosylation of purified bovine DPP9. The purified DPP9 was activated and stabilized by DTT. Bovine DPP9 lost its activity almost completely after alkylation with N-ethylmaleimide. Also alkylation with iodoacetamide inhibited DPP9, albeit only 70%. Other properties of bovine DPP9 are reported, including functional stability and sensitivity towards metal ions. Our results indicate that the short form of DPP9 can be isolated from bovine testes and that it behaves as a stable enzyme suitable for further functional and biochemical characterization as well as for inhibitor screening and characterization.

A peptidomics study reveals the impressive antimicrobial peptide arsenal of the wax moth Galleria mellonella

The complete antimicrobial peptide repertoire of Galleria mellonella was investigated for the first time by LC/MS. Combining data from separate trypsin, Glu-C and Asp-N digests of immune hemolymph allowed detection of 18 known or putative G. mellonella antimicrobial peptides or proteins, namely lysozyme, moricin-like peptides (5), cecropins (2), gloverin, Gm proline-rich peptide 1, Gm proline-rich peptide 2, Gm anionic peptide 1 (P1-like), Gm anionic peptide 2, galiomicin, gallerimycin, inducible serine protease inhibitor 2, 6tox and heliocin-like peptide. Six of these were previously known only as nucleotide sequences, so this study provides the first evidence for expression of these genes. LC/MS data also provided insight into the expression and processing of the antimicrobial Gm proline-rich peptide 1. The gene for this peptide was isolated and shown to be unique to moths and to have an unusually long precursor region (495 bp). The precursor region contained other proline-rich peptides and LC/MS data suggested that these were being specifically processed and were present in hemolymph at very high levels. This study shows that G. mellonella can concurrently release an impressive array of at least 18 known or putative antimicrobial peptides from 10 families to defend itself against invading microbes.

Complete sequencing of GABAA receptor subunit β3 by a rapid technique following in‐gel digestion of the protein

Knowledge on complete sequences is pivotal to identify splice variants, generate specific antibodies and predict conformation. A simple analytical approach to obtain 100% sequence coverage, however, is currently not available. Recombinant gamma-aminobutyric acid A receptor subunits were from insect SF9 cells that were co-transfected with rat alpha1 and His-tag beta 3. The complex of these two subunits was run on blue-native PAGE, followed by multidimensional electrophoretic steps. Spots resolved at the third electrophoretic step were in-gel digested with trypsin, chymotrypsin and Asp-N. In-gel modification of lysines by acetylation was carried out to increase sequence coverage. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using both, collision-induced dissociation and electron transfer dissociation principles. When results from trypsin, chymotrypsin and Asp-N digestion were combined, a single peptide [424 KKTHLRRRSSQL 435] was still not identified. In-gel lysine acetylation leads to unambiguous identification of this peptide by the use of MASCOT v2.2. The use of the Modiro software along with MASCOT, however, was able to provide 100% sequence coverage even without the use of in-gel lysine acetylation. It was observed that the use of trypsin, chymotrypsin and Asp-N with bioinformatic handling by the MASCOT and Modiro software is sufficient to obtain complete sequencing of a highly hydrophobic membrane protein.

Individual b2 ion fragmentation profiles combined with AspN digestion improve N-terminal peptide sequencing

The N terminus of peptides generated by AspN is restricted to about 40 dipeptide motifs starting with D or E. These motifs are visible upon collision-induced dissociation (CID) as b(2) ions, which are often the most abundant low-mass fragment ions. It was observed that b(2) ions are accompanied by a set of sequence-specific neutral losses of CO, H(2)O, NH(3), and some other small units. To test the utility of these profiles as additional parameters for reliable assignment of the b(2) ion motif besides its m/z value, the CID spectra of 221 different AspN-generated peptides covering all N-terminal D-X and E-X motifs were recorded. Qualitatively, the b(2) ion fragmentation profiles of individual motifs were found to exhibit little dependency on the rest of the peptide sequence. Thus, it is concluded that the set of b(2) ion fragmentation profiles recorded in this study can be used as reference set. Knowledge of these profiles provides an increased specificity for b(2) ion annotation of AspN-generated peptides compared to the use of only a solitary b(2) ion m/z value. Recognition of the b(2) ion motif provides a two-amino-acid sequence including its direction; it provides the location of this motif at the N terminus, and it sets a starting point for further extension of the b ion series.

Identification of the Major Phosphorylation Site in Bcl-xL Induced by Microtubule Inhibitors and Analysis of Its Functional Significance

Vinblastine and other microtubule inhibitors used as antimitotic cancer drugs characteristically promote the phosphorylation of the key anti-apoptotic protein, Bcl-xL. However, putative sites of phosphorylation have been inferred based on potential recognition by JNK, and no direct biochemical analysis has been performed. In this study we used protein purification and mass spectrometry to identify Ser-62 as a single major site in vivo. Site-directed mutagenesis confirmed Ser-62 to be the site of Bcl-xL phosphorylation induced by several microtubule inhibitors tested. Vinblastine-treated cells overexpressing a Ser-62 --> Ala mutant showed highly significantly reduced apoptosis compared with cells expressing wild-type Bcl-xL. Co-immunoprecipitation revealed that phosphorylation caused wild-type Bcl-xL to release bound Bax, whereas phospho-defective Bcl-xL retained the ability to bind Bax. In contrast, phospho-mimic (Ser-62 --> Asp) Bcl-xL exhibited a reduced capacity to bind Bax. Functional tests were performed by transiently co-transfecting Bax in the context of different Bcl-xL mutants. Co-expression of wild-type or phospho-defective Bcl-xL counteracted the adverse effects of Bax expression on cell viability, whereas phospho-mimic Bcl-xL failed to provide the same level of protection against Bax. These studies suggest that Bcl-xL phosphorylation induced by microtubule inhibitors plays a key pro-apoptotic role at least in part by disabling the ability of Bcl-xL to bind Bax.

A novel disulphide switch mechanism in Ero1α balances ER oxidation in human cells

Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox-driven shutdown mechanism of Ero1alpha, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1alpha in cells arises from the formation of a disulphide bond-identified by mass spectrometry--between the active-site Cys(94) (connected to Cys(99) in the active enzyme) and Cys(131). Competition between substrate thiols and Cys(131) creates a feedback loop where activation of Ero1alpha is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1alpha-Cys131Ala or the isoform Ero1beta, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis.

Sequence microheterogeneity of parvalbumin pI 5.0 of pike: A mass spectrometric study

Parvalbumin (PA) is a muscle and neuronal calcium-binding protein, the major fish and frog allergen. Its characteristic feature is the presence of multiple isoforms with significantly different amino acid sequences. Here we show that the major isoform of northern pike muscle PA (pI 5.0, α-PA) exhibits microheterogeneity of amino acid sequence. ESI Q-TOF mass-spectrometry (MS) analysis of α-PA sample showed the presence of two components with mass difference of 71 Da. Analysis of tryptic and endoproteinase Asp-N digests of α-PA by MALDI-TOF MS revealed peptides, corresponding to two different amino acid sequences. The sequence differences between variant proteins are limited to AB-domain and include substitutions K27A and L31K, and an extra Leu residue between K11 and K12. Since the affected residues comprise a cluster on the surface of PA, an involvement of the identified region into target recognition is suggested. The substitutions at positions 27 and 31 are located in the region of previously identified epitopes of parvalbumin relevant for PA-specific IgE and IgG binding, which suggests different immunoactivities of the variants. The found microheterogeneity of PA is suggested to be of importance for physiological adaptation of the propulsive musculature to developmental and/or environmental requirements and may contribute to PA allergenicity.

Complementary Structural Mass Spectrometry Techniques Reveal Local Dynamics in Functionally Important Regions of a Metastable Serpin

Serpins display a number of highly unusual structural properties along with a unique mechanism of inhibition. Although structures of numerous serpins have been solved by X-ray crystallography, little is known about the dynamics of serpins in their inhibitory active conformation. In this study, two complementary structural mass spectrometry methods, hydroxyl radical-mediated footprinting and hydrogen/deuterium (H/D) exchange, were employed to highlight differences between the static crystal structure and the dynamic conformation of human serpin protein, alpha(1)-antitrypsin (alpha(1)AT). H/D exchange revealed the distribution of flexible and rigid regions of alpha(1)AT, whereas footprinting revealed the dynamic environments of several side chains previously identified as important for the metastability of alpha(1)AT. This work provides insights into the unique structural design of alpha(1)AT and improves our understanding of its unusual inhibition mechanism. Also, we demonstrate that the combination of the two MS techniques provides a more complete picture of protein structure than either technique alone.

Effects of 4-hydroxynonenal on mitochondrial 3-hydroxy-3-methylglutaryl (HMG-CoA) synthase

Chronic ethanol consumption causes increased production of reactive oxygen species in hepatic mitochondria accompanied by elevations in products of lipid peroxidation such as 4-hydroxynonenal (4-HNE). In the current study we investigated the effects of chronic ethanol consumption on a prominent protein–4-HNE adduct in liver mitochondria. Male Sprague-Dawley rats were fed a liquid diet for 31 days in which ethanol constituted 36% of total calories. Immunoblot analyses of liver mitochondria from ethanol-fed and control animals, using an antibody to a 4-HNE–protein adduct, demonstrated elevated 4-HNE binding (+ 50%) to a mitochondrial protein of ∼ 55 kDa due to chronic ethanol consumption. Analysis of this protein using AspN digestion and tandem mass spectrometry identified it as the mitochondrial form of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. Activity of the activated form of this enzyme was unchanged in livers from ethanol-fed animals, but the protein level was elevated by 36%, which suggests a compensatory mechanism to maintain constant levels of synthase activity in the mitochondrion in the face of continuous inactivation by 4-HNE. Treatment of isolated mitochondria with 4-HNE demonstrated that the enzyme activity decreased as a function of 4-HNE concentration and with time of exposure. This study demonstrates that ethanol consumption increases the formation of a 4-HNE adduct with mitochondrial HMG-CoA synthase, which has the potential to inactivate the enzyme in situ.

Effects of Spontaneous Deamidation on the Cytotoxic Activity of the Bacillus anthracis Protective Antigen

Protective antigen (PA) is a central virulence factor of Bacillus anthracis and a key component in anthrax vaccines. PA binds to target cell receptors, is cleaved by the furin protease, self-aggregates to heptamers, and finally internalizes as a complex with either lethal or edema factors. Under mild room temperature storage conditions, PA cytotoxicity decreased (t(1/2) approximately 7 days) concomitant with the generation of new acidic isoforms, probably through deamidation of Asn residues. Ranking all 68 Asn residues in PA based on their predicted deamidation rates revealed five residues with half-lives of <60 days, and these residues were further analyzed: Asn10 in the 20-kDa region, Asn162 at P6 vicinal to the furin cleavage site, Asn306 in the pro-pore translocation loop, and both Asn713 and Asn719 in the receptor-binding domain. We found that PA underwent spontaneous deamidation at Asn162 upon storage concomitant with decreased susceptibility to furin. A panel of model synthetic furin substrates was used to demonstrate that Asn162 deamidation led to a 20-fold decrease in the bimolecular rate constant (k(cat)/Km) of proteolysis due to the new negatively charged residue at P6 in the furin recognition sequence. Furthermore, reduced PA cytotoxicity correlated with a decrease in PA cell binding and also with deamidation of Asn713 and Asn719. On the other hand, neither deamidation of Asn10 or Asn306 nor impairment of heptamerization could be observed upon prolonged PA storage. We suggest that PA inactivation during storage is associated with susceptible deamidation sites, which are intimately involved in both mechanisms of PA cleavage by furin and PA-receptor binding.

Independent Validation of Candidate Breast Cancer Serum Biomarkers Identified by Mass Spectrometry

We previously selected a panel of 3 breast cancer biomarkers (BC1, BC2, and BC3) from serum samples collected at a single hospital based on their collective contribution to the optimal separation of breast cancer patients and noncancer controls by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). The identities and general applicability of these markers, however, were unknown. In this study, we performed protein expression profiling on samples obtained from a second hospital, included a greater number of ductal carcinoma in situ (DCIS) cases, and performed purification and identification of the 2 confirmed markers. Using a case-control study design, we performed protein expression profiling on serum samples from the National Cancer Institute (Milan, Italy). The validation sample cohort consisted of 61 women with locally invasive breast cancer, 32 with DCIS, 37 with various benign breast diseases (including 13 atypical), and 46 age-matched apparently healthy women (age range, 44-68 years). Validated biomarkers were purified and identified with serial chromatography, 1-dimensional gel electrophoresis, in-gel ASP-N digestion, peptide mass fingerprinting, and tandem mass peptide sequencing. The BC3 and BC2 expression patterns in this sample set were consistent with the first study sample set. BC3 and BC2 were identified to be complement component C3a(desArg) and a C-terminal-truncated form of C3a(desArg), respectively. Evaluation of biomarkers in independent sample sets can help determine the broader utility of candidate markers, and protein identification permits understanding of their molecular basis. C3a(desArg) appears to lack specificity among patients with benign diseases, limiting its utility as a stand-alone tumor marker, but it may still be useful in a multimarker panel for early detection of breast cancer.

Structural characterization of the maytansinoid–monoclonal antibody immunoconjugate, huN901–DM1, by mass spectrometry

Immunoconjugates are being explored as novel cancer therapies with the promise of target-specific drug delivery. The immunoconjugate, huN901-DM1, composed of the humanized monoclonal IgG1 antibody, huN901, and the maytansinoid drug, DM1, is being tested in clinical trials to treat small cell lung carcinoma (SCLC). huN901-DM1 contains an average of three to four DM1 drug molecules per huN901 antibody molecule. The drug molecules are linked to huN901 through random modification of huN901 at epsilon-amino groups of lysine residues, thus yielding a heterogeneous population of conjugate species. We studied the drug distribution profile of huN901-DM1 by electrospray time-of-flight mass spectrometry(ESI-TOFMS), which showed that one to six DM1 drug molecules were attached to an antibody molecule. Both light and heavy chains contained linked drugs. The conjugation sites in both chains were determined by peptide mapping using trypsin and Asp-N protease digestion. Trypsin digestion identified modified lysine residues, since these residues were no longer susceptible to enzymatic cleavage after conjugation with the drug. With respect to Asp-N digestion, modified peptides were identified by observing a mass increase corresponding to the modification. The two digestion methods provided consistent results, leading to the identification of 20 modified lysine residues in both light and heavy chains. Each lysine residue was only partially modified. No conjugation sites were found in complementarity determining regions (CDRs). Using structural models of human IgG1, it was found that modified lysine residues were on the surface in areas of structural flexibility and had large solvent accessibility.

Alkylation of β-Tubulin on Glu 198 by a Microtubule Disrupter

We have shown that beta-tubulin was alkylated by a microtubule disrupter, N-4-iodophenyl-N'-(2-chloroethyl)urea (ICEU), on a glutamic acid residue at position 198 and not on the previously proposed reactive cysteine 239. ICEU belongs to the 4-substituted-phenyl-N'-(2-chloroethyl) urea class that alkylates mainly cellular proteins. Previous studies have shown that the tert-butyl (tBCEU) and iodo (ICEU) derivatives induce microtubule disruption because of beta-tubulin alkylation. tBCEU was supposed to bind covalently to cysteine 239 of beta-tubulin, but this binding site was not clearly confirmed (Cancer Res 60:985-992, 2000). We have isolated and analyzed beta-tubulin after two-dimensional gel electrophoresis of proteins from B16 cells incubated with ICEU. Alkylated beta-tubulin had a lower apparent molecular weight and a more basic isoelectric point than the unmodified protein. Labeled N-4-[125I]CEU was effectively bound to the modified beta-tubulin but using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, we demonstrated that none of the cysteine residues of beta-tubulin was linked to the alkylating agent. In contrast, peptide masses at m/z 4883 and 1792 in trypsin or Asp-N digestions of beta-tubulin confirmed binding of iodophenylethylureido moiety to peptides [175-213] or [197-208] respectively. Fragmentation analyses by electrospray mass spectrometry using triply charged ions of peptide [175-213] identified a glutamic acid at position 198 as target for alkylation via an ester bond with ICEU. This amino acid located in the intermediate domain of the beta-tubulin should play an essential role in the conformational structure necessary for the interaction between dimers in the protofilament.