Analysis of TPOX short tandem repeat locus with matrix-associated laser desorption/ionization time-of-flight-based restriction fragment mass polymorphism assay

Short tandem repeat (STR) loci are routinely analyzed by capillary electrophoresis. However, this method has several disadvantages, including long operational time, low throughput, and inaccuracy. As a result of the introduction of matrix-associated laser desorption/ionization time-of-flight (MALDI–TOF) and electrospray ionization (ESI), mass spectrometry has become an alternative method for genotyping polymorphic STR loci. Here we established a restriction fragment mass polymorphism (RFMP) assay for genotyping STR locus, TPOX, by typeIIS restriction endonuclease cleavage of polymerase chain reaction (PCR) amplicon followed by MALDI–TOF mass spectrometry. The resulting TPOX genotypes from this assay were in good agreement with the results from direct DNA sequencing and GeneScan assays. Our results showed that the RFMP assay is an accurate and high-throughput method for analyzing long DNA fragments such as STR markers. Further research with multiple STR loci may allow this assay to be used for diverse applications such as forensics, paternity tests, and detection of genetic disorders.     

Quantification of periviscerokinin‐1 in the nervous system of the American cockroach,Periplaneta americana: An insect neuropeptide with unusual distribution

This study was undertaken to reveal the quantitative distribution of the insect neuropeptide periviscerokinin‐1 (Pea‐PVK‐1) in the central nervous system of Periplaneta americana and to demonstrate that neurons stained in a previous immunohistochemical study contain authentic Pea‐PVK‐1. For this, we combined ELISA, HPLC, and MALDI‐TOF mass spectrometry. The high specificity of the used antiserum enabled the quantification of Pea‐PVK‐1 in unseparated tissue extracts. No cross‐reactivities with other insect neuropeptides were detected in ELISA. Only two immunoreactive fractions, coeluting with synthetic Pea‐PVK‐1 in its oxidized and nonoxidized form, were found in HPLC‐separated extracts of the brain, suboesophageal ganglion, metathoracic ganglion, second abdominal ganglion with or without perisympathetic organ, and terminal ganglion. By using MALDI‐TOF mass spectrometry, we were able to confirm the existence of authentic Pea‐PVK‐1 in these fractions. The abdominal perisympathetic organs contained 6.3 pmol Pea‐PVK‐1 per animal; another 1.3 pmol were found in the abdominal ganglia. More than 90% of the total 8.2 pmol in the central nervous system was found in the abdominal ganglia and their perisympathetic organs. The corpora cardiaca and corpora allata did not contain immunoreactive material, suggesting that Pea‐PVK‐1 is not released by the cephalic neurohaemal system. The quantitative distribution of Pea‐PVK‐1 differs considerably from that of other known insect neuropeptides. Arch. Insect Biochem. Physiol. 40:203–211, 1999. © 1999 Wiley‐Liss, Inc.     

Chemical synthesis and structural characterization of the RGD-protein decorsin: a potent inhibitor of platelet aggregation.

Decorsin is a 39-residue RGD-protein crosslinked by three disulfide bridges isolated from the leech Macrobdella decora belonging to the family of GPIIb-IIIa antagonists and acting as a potent inhibitor of platelet aggregation. Here we report the solid-phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse-phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse-phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synthetic protein, NH2-terminal sequencing and mass determination (4,377 Da) by electrospray mass spectrometry were in full agreement with this theory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mapping using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near-UV region. Synthetic decorsin inhibited human platelet aggregation with an IC50 of approximately 0.1 microM, a figure quite similar to that determined utilizing decorsin from natural source. In particular, the synthetic protein was 2,000-fold more potent than a model RGD-peptide (e.g., Arg-Gly-Asp-Ser) in inhibiting platelet aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (Tm approximately 74 degrees C). The features of the oxidative refolding process of reduced decorsin, as well as the thermal stability of the oxidized species, were compared with those previously determined for the NH2-terminal core domain fragment 1-41 or 1-43 from hirudin. This fragment shows similarity in size, pairing of the three disulfides and three-dimensional structure with those of decorsin, even if very low sequence similarity. It is suggested that the less efficient oxidative folding and the enhanced thermal stability of decorsin in respect to those of hirudin core domain likely can be ascribed to the presence of the six Pro residues in the decorsin chain, whereas none is present in the hirudin domain. The results of this study indicate that decorsin can be obtained by solid-phase methodology in purity and quantities suitable for structural and functional studies and thus open the way to prepare by chemical methods novel decorsin derivatives containing unusual amino acids or even non-peptidic moieties.     

Identification and structure of a putative Ca2+-binding domain at the C terminus of AQP1

Aquaporin-1 (AQP1) is the first functionally identified aquaporin of a growing family of membrane water channels found in all forms of life. Recently, a possible secondary function as a cyclic guanosine monophosphate (cGMP) gated ion channel was attributed to AQP1. We have reconstituted purified protein from bovine and human red blood cell membranes into highly ordered 2D crystals. The topography of both AQP1s was determined by electron microscopy from freeze-dried, unidirectionally metal-shadowed 2D crystals as well as from surface topographs of native crystals recorded in buffer solution with the atomic force microscope (AFM). In spite of the high level of sequence homology between bovine and human AQP1, the surfaces showed distinct differences. Alignment of both sequences and comparison of the acquired surface topographies with the atomic model of human AQP1 revealed the topographic changes on the surface of bovine AQP1 to be induced by a few amino acid substitutions. A striking degree of sequence homology was found between the carboxyl-terminal domains of AQP1s from different organisms and EF-hands from Ca2+-binding proteins belonging to the calmodulin superfamily, suggesting the existence of a Ca2+-binding site at the C terminus of AQP1 instead of the putative cGMP-binding site reported previously. To unveil its position on the acquired surface topographies, 2D crystals of AQP1 were digested with carboxypeptidase Y, which cleaves off the intracellular C terminus. Difference maps of AFM topographs between the native and the peptidase-treated AQP1s showed the carboxylic tail to be close to the 4-fold symmetry axis of the tetramer. SDS-PAGE and matrix-assisted laser desorption/ionisation mass spectrometry of native and decarboxylated bovine and human AQP1 revealed that the EF-hand motif found at the C terminus of AQP1 was partially resistant to peptidase digestion. The importance of the C-terminal domain is implicated by structural instability of decarboxylated AQP1. A possible role of the C terminus and calcium in translocation of AQP1 in cholangiocytes from intracellular vesicles to the plasma membrane and in triggering its fusion is discussed. Functional studies are now required to identify the physiological role of the Ca2+-binding site.     

Intraneuronal amyloid-beta1-42 production triggered by sustained increase of cytosolic calcium concentration induces neuronal death

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence in the brain of senile plaques which contain an amyloid core made of beta-amyloid peptide (Abeta). Abeta is produced by the cleavage of the amyloid precursor protein (APP). Since impairment of neuronal calcium signalling has been causally implicated in ageing and AD, we have investigated the influence of an influx of extracellular calcium on the metabolism of human APP in rat cortical neurones. We report that a high cytosolic calcium concentration, induced by neuronal depolarization, inhibits the alpha-secretase cleavage of APP and triggers the accumulation of intraneuronal C-terminal fragments produced by the beta-cleavage of the protein (CTFbeta). Increase in cytosolic calcium concentration specifically induces the production of large amounts of intraneuronal Abeta1-42, which is inhibited by nimodipine, a specific antagonist of l-type calcium channels. Moreover, calcium release from endoplasmic reticulum is not sufficient to induce the production of intraneuronal Abeta, which requires influx of extracellular calcium mediated by the capacitative calcium entry mechanism. Therefore, a sustained high concentration of cytosolic calcium is needed to induce the production of intraneuronal Abeta1-42 from human APP. Our results show that this accumulation of intraneuronal Abeta1-42 induces neuronal death, which is prevented by a functional gamma-secretase inhibitor.     

Evidence of different G-quadruplex DNA binding with biogenic polyamines probed by electrospray ionization-quadrupole time of flight mass spectrometry,circular dichroism and atomic force microscopy

The binding properties of five G-quadruplex oligonucleotides (humtel24, k-ras32, c-myc22, c-kit1 and c-kit2) with polyamines have been investigated by electrospray ionization-quadrupole time of flight mass spectrometry, circular dichroism, melting temperature, atomic force microscopy (AFM) and molecular simulation. The MS results demonstrated that the polyamines and G-quadruplex DNA can form complexes with high affinity, and one molecule of G-quadruplex DNA can combine several molecules (1–5) of polyamines. The binding affinities of the polyamines to DNA were in the order of spermine > spermidine > putrescine. After binding with polyamines, the conformations of the G-quadruplex DNA were significantly changed, and spermine can induce the configurations of k-ras32 and c-kit1 to deviate from their G-quadruplex structures at high concentrations. In the presence of K⁺, the conformations of G-quadruplex DNA were stabilized, while polyamines can also induced alterations of their configurations. Melting temperature experiments suggested that the T_m of the DNA–polyamine complexes obviously increased both in the absence and presence of K⁺. The AFM results indicated that polyamines can induce aggregation of G-quadruplex DNA. Above results illustrated that the polyamines bound with the phosphate backbone and the base-pairs of G-quadruplex structures. Combining with the molecular simulation, the binding mode of the G-quadruplex DNA and polyamines were discussed. The results obtained would be beneficial for understanding the biological and physiological functions of polyamines and provide useful information for development of antitumor drugs.     

Enhanced G-protein activation by a mixture of Abeta(25-35), Abeta(1-40/42) and zinc

Beta-amyloid peptides (Abetas) bind to several G-protein coupled receptor proteins and stimulate GTPase activity in neurons. In this study we determined the effects of Abeta(1-42), Abeta(1-40), Abeta(25-35) and their mixtures on [(35)S]GTP binding in rat brain cortical membranes in the absence and presence of zinc. We found that the Abetas alone induced a concentration-dependent activation of G-proteins (IC50 approximately 10(-6) m), while aggregated Abeta fibrils only affected GTP binding at concentrations above 10(-5) m. Mixing Abeta(25-35) with Abeta(1-42) or Abeta(1-40) induced a several-fold increase in GTP-binding. This potentiation followed a bell shaped curve with a maximum at 50 : 50 ratios. No potentiating effect could be seen by mixing Abeta(1-40) and Abeta(1-42) or highly aggregated Abetas. Zinc had no effect on Abeta(1-40/42) but strongly potentiated the Abeta(25-35) or the mixed peptides-induced GTP-binding. Changes in secondary structure accompanied the mixed peptides or the peptide/zinc complexes induced potentiation, revealing that structural alterations are behind the increased biological action. These concentration dependent potentiating effects of zinc and the peptide mixtures could be physiologically important at brain regions where peptide fragments and/or zinc are present at elevated concentrations.     

Surface morphology and chemistry of<Emphasis Type="Italic"> Prunus laurocerasus</Emphasis> L. leaves: a study using X-ray photoelectron spectroscopy,time-of-flight secondary-ion mass spectrometry,atomic-force microscopy and scanning-electron microscopy

The surface properties of the plant cuticle play a crucial role in plant–pathogen interactions and the retention and penetration of agriculturally important chemicals. This paper describes the use of X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (ToF-SIMS), tapping-mode atomic force microscopy (TM-AFM) and scanning electron microscopy (SEM) to determine surface-specific chemical and material properties of the adaxial surface of Prunus laurocerasus L. leaves. XPS data, derived from the uppermost few nanometres (<10 nm) of the leaf surface, were consistent with the wax components and functionality known to be present within the waxes. ToF-SIMS provided molecular speciation from the outermost monolayer of the leaf surface, indicating the importance of a family of acetates with chain lengths ranging from C₂₀ to C₃₄. The presence of alkanes with C₂₉ and C₃₁ chain lengths was also confirmed. SEM and TM-AFM topography images revealed a textured granular surface, while simultaneously recorded AFM phase images revealed heterogeneous material properties at the nanoscale. The relevance of these data to plant cuticle development, allelochemistry and agrochemical delivery is discussed.     

Characterization of a Yersinia enterocolitica biotype 1A strain harbouring an ail gene

Aims: The chromosomal ail gene (attachment and invasion locus) is commonly used as target gene for the detection of pathogenic Y. enterocolitica strains in food testing. The ail PCR does not detect strains of biotype 1A (BT1A), which are regarded as non‐pathogenic because BT1A strains lack the virulence plasmid and chromosomally encoded virulence genes. In some recent reports, however, BT1A strains were discovered that harboured the ail gene. We isolated an ail‐positive strain and characterized this strain with phenotypic and genotypic methods to study its possible relation to pathogenic Y. enterocolitica strains. Methods and Results: The ail region of the BT1A strain was sequenced and compared with the corresponding region of nonpathogenic BT1A strains and pathogenic strains. Pulsed field gel electrophoresis (PFGE) analysis was applied revealing no similarity of the PFGE pattern of this strain to the patterns of pathogenic strains. Virulence‐gene‐based PCR analyses showed the strain to be positive for ystB, but negative for virulence genes ystA, virF and yadA. Whole‐cell MALDI‐TOF MS combined with a shrinkage discriminant analysis approach was applied and clearly classified the ail‐positive biotype 1A strain within the cluster of BT1A strains. Conclusions: PCR detection of ail sequences in food matrices should be followed by the isolation of the responsible strain and its characterization using phenotypic or genotypic methods. Significance and Impact of the Study: The ail gene may be present in Y. enterocolitica BT1A strains, which are commonly considered as nonpathogenic. Efficient methods such as PCR typing of other virulence genes or rapid MALDI‐TOF MS‐based bacterial profiling allow a more comprehensive assessment of the pathogenicity potential of Yersinia strains.     

Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans

Mannose-6-phosphate (M-6-P) glycan analysis is important for quality control of therapeutic enzymes for lysosomal storage diseases. Here, we found that the analysis of glycans containing two M-6-Ps was highly affected by the hydrophilicity of the elution solvent used in high-performance liquid chromatography (HPLC). In addition, the performances of three fluorescent tags—2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), and 3-(acetyl-amino)-6-aminoacridine (AA-Ac)—were compared with each other for M-6-P glycan analysis using HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The best performance for analyzing M-6-P glycans was shown by 2-AA labeling in both analyses.     

Amyloid β (1-42) Folding Multiplicity and Single-Molecule Binding Behavior Studied with STM

The fine folding and assembling characteristics of amyloid β (Aβ) peptides are important to pharmaceutical studies of drug molecules and to the pathological analysis of neurodegenerative disorders such as Alzheimer's disease at the molecular level. Here we present observations of the multiple folding characteristics of amyloid peptide Aβ₄₂ lamellae using scanning tunneling microscopy. Molecularly resolved core regions of Aβ₄₂ hairpins and unfolded peptide assembly structures are identified. The parallel assembling characteristics of Aβ₄₂ hairpins can be confirmed in the study. In addition, single-molecule binding characteristics of Congo red and thioflavin T have been shown to bind at the groove regions of peptide assemblies. This study demonstrates a complementary venue for studying molecular heterogeneity of peptide assemblies, as well as the binding characteristics of molecular modulators.     

Spontaneous aggregation and cytotoxicity of the beta-amyloid Abeta1-40: a kinetic model

The time dependency of the spontaneous aggregation of the fibrillogenic -Amyloid peptide, A^1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-A antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 M amyloid peptide. The anti–A antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length A was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the A^1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of A^1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of A^1–40 in vitro and possibly in vivo.     

Characterization of copper binding to the peptide amyloid-beta(1-16) associated with Alzheimer's disease

Amyloid-beta peptide (Abeta) is the principal constituent of plaques associated with Alzheimer's disease (AD) and is thought to be responsible for the neurotoxicity associated with the disease. Copper binding to Abeta has been hypothesized to play an important role in the neruotoxicity of Abeta and free radical damage, and Cu2+ chelators represent a possible therapy for AD. However, many properties of copper binding to Abeta have not been elucidated clearly, and the location of copper binding sites on Abeta is also in controversy. Here we have used a range of spectroscopic techniques to characterize the coordination of Cu2+ to Abeta(1-16) in solution. Electrospray ionization mass spectrometry shows that copper binds to Abeta(1-16) at pH 6.0 and 7.0. The mode of copper binding is highly pH dependent. Circular dichroism results indicate that copper chelation causes a structural transition of Abeta(1-16). UV-visible absorption spectra suggest that three nitrogen donor ligands and one oxygen donor ligand (3N1O) in Abeta(1-16) may form a type II square-planar coordination geometry with Cu2+. By means of fluorescence spectroscopy, competition studies with glycine and L-histidine show that copper binds to Abeta(1-16) with an affinity of Ka approximately 10(7) M(-1) at pH 7.8. Besides His6, His13, and His14, Tyr10 is also involved in the coordination of Abeta(1-16) with Cu2+, which is supported by 1H NMR and UV-visible absorption spectra. Evidence for the link between Cu2+ and AD is growing, and this work has made a significant contribution to understanding the mode of copper binding to Abeta(1-16) in solution.     

A new method of biosensing with 1 microl of Escherichia coli suspension using atomic force microscopy

We developed a new method for detecting bacterial cells from 1-mul samples with atomic force microscopy (AFM). The use of a parafilm surface as a sample palette was effective for reacting small amounts of samples with an AFM probe. This was due to the parafilm's hydrophobic, semitransparent, and nonadhesive surface. In this way, all processes, such as the surface functionalization of a cantilever and the adhesion of Escherichia coli cells to a cantilever, were easily completed. In addition, we succeeded in detecting cell adsorption on the same AFM cantilever by both the drive mode and the thermal mode. The resonance frequency shift caused by cell adhesion was clearly detected by the two modes for the first time. Our data indicated the potential of applying AFM nanobiosensing to extremely small amounts of samples.     

On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format

Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography–mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins.     

Tau protein binding forms a 1 nm thick layer along protofilaments without affecting the radial elasticity of microtubules

Tau is one of the most abundant microtubule-associated proteins involved in kinetic stabilization and bundling of axonal microtubules. Although intense research has revealed much about tau function and its involvement in Alzheimer's disease during the past years, it still remains unclear how exactly tau binds on microtubules and if the kinetic stabilization of microtubules by tau is accompanied, at least in part, by a mechanical reinforcement of microtubules. In this paper, we have used atomic force microscopy to address both aspects by visualizing and mechanically analyzing microtubules in the presence of native tau isoforms. We could show that tau at saturating concentrations forms a 1 nm thick layer around the microtubule, but leaves the protofilament structure well visible. The latter observation argues for tau binding mainly along and not across the protofilaments. The radial elasticity of microtubules was almost unaffected by tau, consistent with tau binding along the tops of the protofilaments. Tau did increase the resistance of microtubules against rupture. Finite-element calculations confirmed our findings.     

Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129.

Aspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed-phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126-133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides.     

Synthesis and structural characterization of 1-(D-glycosyloxy)phthalazines

Coupling of the trimethylsilyl derivative of (2H)phthalazin-1-one with 1,2,3,4,6-penta-O-acetyl-alpha-D-glucopyranose and 1,2,3,4,6-penta-O-acetyl-alpha-D-galactopyranose in the presence of stannic chloride gave the respective glycosides, 2-(per-O-acetyl-D-glycosyloxy)phthalazines, which upon deacetylation gave the respective unprotected analogues. Under the same conditions 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose gave 1-(2,3,5-tri-O-acetyl-alpha-D-ribofuranosyloxy)phthalazine. Electrospray mass spectrometry aided the structural characterization of this series of 1-(D-glycosylyloxy)phthalazines. Low energy collisionally-induced dissociation tandem mass spectrometry of the protonated molecules confirmed the MS fragmentation routes and the structural identities of this novel series of glycosides.     

Formation of monoclonal antibody against a major ginseng component, ginsenoside Rb1 and its characterization

The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibody against ginsenoside Rb1 was produced by fusing splenocytes immunized with a ginsenoside Rb1- bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A very small cross-reaction appeared with ginsenoside Rc and ginsenoside Rd. The full measuring range of the assay extends from 20 ngml-1 to 400 ngml-1 of ginsenoside Rb1. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins.