RESUMO
The higher order structure (HOS) of proteins plays a critical role in the efficacy and stability of biological drugs. Perturbation of the regional structure of proteins can affect biological activity and cause instability. Characterization of HOS has become an integral part of biological drug development and is expected from regulatory agencies. The commonly used techniques for HOS characterization, such as circular dichroism, Fourier-transform infrared, differential scanning calorimetry, intrinsic fluorescence, and hydrogen-deuterium exchange mass spectrometry, have their limitations ranging from lack of sensitivity and specificity to the need of high-level expertise and poor access to instrumentation due to high cost. In this study, we demonstrated a novel controlled proteolysis-based LC-QDa method for the detection of HOS change. By digesting proteins directly without denaturation and reduction, the HOS information can be revealed through the digested peptides. After optimizing the digestion conditions and the detection procedures, we identified 13 signature peptides that can monitor various antibody domains for any HOS changes caused by external stress. By comparing the peptide peak areas between unknown samples and a native control sample, any regional structural changes in unknown samples can be detected. The method was subsequently applied to a wide range of forced degradation samples to demonstrate higher sensitivity compared to the near-UV CD method that is frequently used for monitoring tertiary structural changes. By further reducing the number of signature peptides to five and optimizing liquid chromatography gradient duration, a streamlined, high-throughput, and controlled proteolysis method was successfully established. This method can be used to support process and formulation development as well as potentially for stability testing.
Assuntos
Proteínas/química , Modelos Moleculares , Conformação Proteica , ProteóliseRESUMO
MnO2 nanoparticles, similar to those found in soils and sediments, have been characterized via their UV-visible and Raman spectra, combined with dynamic light scattering and reactivity measurements. Synthetic colloids were prepared by thiosulfate reduction of permanganate, their sizes controlled with adsorbates acting as capping agents: bicarbonate, phosphate, and pyrophosphate. Biogenic colloids, products of the manganese oxidase, Mnx, were similarly characterized. The band-gap energies of the colloids were found to increase with decreasing hydrodynamic diameter, Dh, and were proportional to 1/ Dh2, as predicted from quantum confinement theory. The intensity ratio of the two prominent Mn-O stretching Raman bands also varied with particle size, consistent with the ratio of edge to bulk Mn atoms. Reactivity of the synthetic colloids toward reduction by Mn2+, in the presence of pyrophosphate to trap the Mn3+ product, was proportional to the surface to volume ratio, but showed surprising complexity. There was also a remnant unreactive fraction, likely attributable to Mn(III)-induced surface passivation. The band gap was similar for biogenic and synthetic colloids of similar size, but decreased when the enzyme solution contained pyrophosphate, which traps the intermediate Mn(III) and slows MnO2 growth. The band gap/size correlation was used to analyze the growth of the enzymatically produced MnO2 oxides.
Assuntos
Compostos de Manganês , Nanopartículas , Difusão Dinâmica da Luz , Manganês , Óxidos , Oxirredutases , Tamanho da PartículaRESUMO
Methionine oxidation is a major degradation pathway in therapeutic proteins which can impact the structure and function of proteins as well as risk to drug product quality. Detecting Met oxidation in proteins by peptide mapping followed by liquid chromatography with mass spectrometry (LC-MS) is the industry standard but is also labor intensive and susceptible to artifacts. In this work, vibrational difference spectroscopy in combination with 18O isotopic shift enabled us to demonstrate the application of Raman and FTIR techniques for the detection and quantification of Met oxidation in various therapeutic proteins, including mAbs, fusion proteins, and antibody drug conjugate. Vibrational markers of Met oxidation products, such as sulfoxide and sulfone, corresponding to SâO and C-SâO stretching frequencies were unequivocally identified based 18O isotoptic shifts. The intensity of the isolated νC-S Raman band at 702 cm-1 was successfully applied to quantify the average Met oxidation level in multiple proteins. These results are further corroborated by oxidation levels measured by tryptic peptide mapping, and thus the confirmed Met oxidation levels derived from Raman and mass spectrometry are indeed consistent with each other. Thus, we demonstrate the broader application of vibrational spectroscopy to detect the subtle spectral changes associated with various chemical or physical degradation of proteins, including Met oxidation as well as higher order structural changes.
Assuntos
Anticorpos Monoclonais/química , Metionina/análise , Proteínas Recombinantes de Fusão/química , Sulfonas/análise , Sulfóxidos/análise , Anticorpos Monoclonais/metabolismo , Biomarcadores/análise , Cromatografia Líquida , Espectrometria de Massas , Metionina/metabolismo , Oxirredução , Proteínas Recombinantes de Fusão/metabolismo , VibraçãoRESUMO
Optically heterodyne-detected femtosecond Raman-induced Kerr-effect spectroscopy (OHD-FRIKES) was observed in neat cyclohexane. In this Letter, an examination of the effect of the Raman pump ellipticity on the multiplex OHD-FRIKES spectra is discussed. The Raman pump ellipticity scanned OHD-FRIKES results reproduce anomalous observables from previous OHD-FRIKES experiments and suggest new methods of tracking transient vibrational mode polarization in complex systems.
RESUMO
Protein dynamics on the microsecond (µs) time scale were investigated by temperature-jump fluorescence spectroscopy as a function of temperature in two variants of a thermophilic alcohol dehydrogenase: W87F and W87F:H43A. Both mutants exhibit a fast, temperature-independent µs decrease in fluorescence followed by a slower full recovery of the initial fluorescence. The results, which rule out an ionizing histidine as the origin of the fluorescence quenching, are discussed in the context of a Trp49-containing dimer interface that acts as a conduit for thermally activated structural change within the protein interior.
Assuntos
Álcool Desidrogenase/química , Espectrometria de Fluorescência/métodos , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Fluorescência , Geobacillus stearothermophilus/enzimologia , Histidina/química , Mutação , Conformação Proteica , Multimerização Proteica , Temperatura , Triptofano/químicaRESUMO
The recently developed technique of femtosecond stimulated Raman spectroscopy, and its variant, femtosecond Raman-induced Kerr effect spectroscopy (FRIKES), offer access to ultrafast excited-state dynamics via structurally specific vibrational spectra. We have used FRIKES to study the photoexcitation dynamics of nickel(II) phthalocyanine with eight butoxy substituents, NiPc(OBu)8. NiPc(OBu)8 is reported to have a relatively long-lived ligand-to-metal charge-transfer (LMCT) state, an essential characteristic for efficient electron transfer in photocatalysis. Following photoexcitation, vibrational transitions in the FRIKES spectra, assignable to phthalocyanine ring modes, evolve on the femtosecond to picosecond time scales. Correlation of ring core size with the frequency of the ν10 (asymmetric C-N stretching) mode confirms the identity of the LMCT state, which has a â¼500 ps lifetime, as well as that of a precursor d-d excited state. An even earlier (â¼0.2 ps) transient is observed and tentatively assigned to a higher-lying Jahn-Teller-active LMCT state. This study illustrates the power of FRIKES spectroscopy in elucidating ultrafast molecular dynamics.
Assuntos
Indóis/química , Níquel/química , Compostos Organometálicos/química , Isoindóis , Ligantes , Estrutura Molecular , Análise Espectral Raman , Fatores de TempoRESUMO
The use of hybrid hemoglobin (Hb), with mesoheme substituted for protoheme, allows separate monitoring of the α or ß hemes along the allosteric pathway. Using resonance Raman (rR) spectroscopy in silica gel, which greatly slows protein motions, we have observed that the Fe-histidine stretching frequency, νFeHis, which is a monitor of heme reactivity, evolves between frequencies characteristic of the R and T states, for both α or ß chains, prior to the quaternary R-T and T-R shifts. Computation of νFeHis, using QM/MM and the conformational search program PELE, produced remarkable agreement with experiment. Analysis of the PELE structures showed that the νFeHis shifts resulted from heme distortion and, in the α chain, Fe-His bond tilting. These results support the tertiary two-state model of ligand binding (Henry et al., Biophys. Chem. 2002, 98, 149). Experimentally, the νFeHis evolution is faster for ß than for α chains, and pump-probe rR spectroscopy in solution reveals an inflection in the νFeHis time course at 3 µs for ß but not for α hemes, an interval previously shown to be the first step in the R-T transition. In the α chain νFeHis dropped sharply at 20 µs, the final step in the R-T transition. The time courses are fully consistent with recent computational mapping of the R-T transition via conjugate peak refinement by Karplus and co-workers (Fischer et al., Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 5608). The effector molecule IHP was found to lower νFeHis selectively for α chains within the R state, and a binding site in the α1α2 cleft is suggested.
Assuntos
Biologia Computacional/métodos , Heme/química , Hemoglobina A/química , Análise Espectral Raman/métodos , alfa-Globinas/química , Globinas beta/química , Regulação Alostérica , Mesoporfirinas/química , Modelos Moleculares , Estrutura Quaternária de ProteínaRESUMO
The gaseous XO molecules (X = C, N or O) bind to the heme prosthetic group of heme proteins, and thereby activate or inhibit key biological processes. These events depend on interactions of the surrounding protein with the FeXO adduct, interactions that can be monitored via the frequencies of the Fe-X and X-O bond stretching modes, νFeX and νXO. The frequencies can be determined by vibrational spectroscopy, especially resonance Raman spectroscopy. Backbonding, the donation of Fe dπ electrons to the XO π* orbitals, is a major bonding feature in all the FeXO adducts. Variations in backbonding produce negative νFeX/νXO correlations, which can be used to gauge electrostatic and H-bonding effects in the protein binding pocket. Backbonding correlations have been established for all the FeXO adducts, using porphyrins with electron donating and withdrawing substituents. However the adducts differ in their response to variations in the nature of the axial ligand, and to specific distal interactions. These variations provide differing vantages for evaluating the nature of protein-heme interactions. We review experimental studies that explore these variations, and DFT computational studies that illuminate the underlying physical mechanisms.
RESUMO
Encapsulation of hemoglobin (Hb) in silica gel preserves structure and function but greatly slows protein motion, thereby providing access to intermediates along the allosteric pathway that are inaccessible in solution. Resonance Raman (RR) spectroscopy with visible and ultraviolet laser excitation provides probes of heme reactivity and of key tertiary and quaternary contacts. These probes were monitored in gels after deoxygenation of oxyHb and after CO binding to deoxyHb, which initiate conformational change in the R-T and T-R directions, respectively. The spectra establish that quaternary structure change in the gel takes a week or more but that the evolution of heme reactivity, as monitored by the Fe-histidine stretching vibration, ν(FeHis), is completed within two days, and is therefore uncoupled from the quaternary structure. Within each quaternary structure, the evolving ν(FeHis) frequencies span the full range of values between those previously associated with the high- and low-affinity end states, R and T. This result supports the tertiary two-state (TTS) model, in which the Hb subunits can adopt high- and low-affinity tertiary structures, r and t, within each quaternary state. The spectra also reveal different tertiary pathways, involving the breaking and reformation of E and F interhelical contacts in the R-T direction but not the T-R direction. In the latter, tertiary motions are restricted by the T quaternary contacts.
Assuntos
Heme/metabolismo , Hemoglobina A/metabolismo , Proteínas Imobilizadas/metabolismo , Análise Espectral Raman/métodos , Monóxido de Carbono/metabolismo , Heme/química , Hemoglobina A/química , Humanos , Proteínas Imobilizadas/química , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Cytochrome c unfolds locally and reversibly upon heating at pH 3. UV resonance Raman (UVRR) spectra reveal that instead of producing unordered structure, unfolding converts turns and some helical elements to ß-sheet. It also disrupts the Met80-heme bond, and has been previously shown to induce peroxidase activity. Aromatic residues that are H-bonded to a heme propionate (Trp59 and Tyr48) alter their orientation, indicating heme displacement. T-jump/UVRR measurements give time constants of 0.2, 3.9, and 67 µs for successive phases of ß-sheet formation and concomitant reorientation of Trp59. UVRR spectra reveal protonation of histidines, and specifically of His26, whose H-bond to Pro44 anchors the 40s Ω loop; this loop is known to be the least stable 'foldon' in the protein. His26 protonation is proposed to disrupt its H-bond with Pro44, triggering the extension of a short ß-sheet segment at the 'neck' of the 40s Ω loop into the loop itself and back into the 60s and 70s helices. The secondary structure change displaces the heme via H-bonds from residues in the growing ß-sheet, thereby exposing it to exogenous ligands, and inducing peroxidase activity. This unfolding mechanism may play a role in cardiolipin peroxidation by cyt c during apoptosis.
Assuntos
Apoptose , Citocromos c/química , Heme/química , Histidina/química , Dicroísmo Circular , Modelos Moleculares , Prótons , Espectrofotometria Ultravioleta , Análise Espectral RamanRESUMO
The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.
Assuntos
Laboratórios , Software , Tamanho da PartículaRESUMO
Raman spectroscopy can provide unique information on the evolution of structure in proteins over a wide range of time scales; the picosecond to millisecond range can be accessed with pump-probe techniques. Specific parts of the molecule are interrogated by tuning the probe laser to a resonant electronic transition, including the UV transitions of aromatic residues and of the peptide bond. Advances in laser technology have enabled the characterization of transient species at an unprecedented level of structural detail. Applications to protein unfolding and allostery are reviewed.
Assuntos
Proteínas/química , Análise Espectral Raman/métodos , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Espalhamento de Radiação , Sensibilidade e Especificidade , Termodinâmica , Raios UltravioletaRESUMO
N-linked glycosylation is an important post translational modification that occurs on Asparagine 297 residue or a homologous position on the Fc portion of monoclonal antibodies (mAbs). mAb Fc glycans play important roles in antibody structure, stability, and function including effector function and pharmacokinetics. The Fc glycans are made up of a wide variety of sugars including galactose, mannose, and sialic acid. The role of galactose in mediating antibody effector functions is not well understood. Hence, there is widespread interest in the antibody research community to understand the role of galactose in antibody effector functions as galactose is a major constituent of antibody glycans. This requires generation of highly enriched galactosylated variants that has been very challenging via cell culture process. To tackle this challenge, we developed a laboratory scale biochemical process to produce highly enriched galactosylated variants. In this article, we report optimized lab-scale workflows and detailed protocols for generation of deglycosylated, hypo-galactosylated and hyper-galactosylated variants of IgG therapeutic antibodies using the in-vitro glycoengineering technology. The optimized workflows offer short turnaround time and produce highly enriched deglycosylated/hypo-galactosylated/hyper-galactosylated IgG glycovariants, with high purity & molecular integrity as demonstrated by data from an example IgG.
Assuntos
Fragmentos Fc das Imunoglobulinas , Laboratórios , Anticorpos Monoclonais/metabolismo , Glicosilação , Fragmentos Fc das Imunoglobulinas/metabolismo , Polissacarídeos , TecnologiaRESUMO
Methionine-rich motifs have an important role in copper trafficking factors, including the CusF protein. Here we show that CusF uses a new metal recognition site wherein Cu(I) is tetragonally displaced from a Met2His ligand plane toward a conserved tryptophan. Spectroscopic studies demonstrate that both thioether ligation and strong cation-pi interactions with tryptophan stabilize metal binding. This novel active site chemistry affords mechanisms for control of adventitious metal redox and substitution chemistry.
Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Metionina/metabolismo , Proteínas de Transporte de Cátions/genética , Cátions/química , Cátions/metabolismo , Cobre/química , Proteínas de Transporte de Cobre , Proteínas de Escherichia coli , Metionina/genética , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Monoclonal antibodies are heterogeneous in nature and may contain numerous variants with differences in size, charge, and hydrophobicity, which may impact clinical efficacy, immunogenicity, and safety. Characterization of antibody variants is necessary to build structure-function correlation and establish a proper control strategy. Isolation and enrichment of variants by conventional chromatographic peak fractionation is labor-intensive and time-consuming. The instability of fractions during isolation and subsequent characterization may also be a concern. Hence, it is desirable to analyze antibody variants in an online and real-time manner. Here we demonstrate a 2D-LC methodology - multiple heart-cutting IEC-SEC- as an investigational tool to facilitate a charge variant characterization study. Both IEC modes - anion exchange (AEX) and cation exchange (CEX) chromatography are discussed. Using this approach, direct bridging of size and charge variants of an antibody molecule was achieved without offline peak fractionation. It was observed that antibody aggregates elute late on both the AEX and CEX columns, presumably due to secondary hydrophobic interactions. Additionally, we overcame the solvent mismatch issue and developed a 2D SEC-IEC method to confirm the bridging results. This is the first reported SEC-IEC 2D-LC application for the characterization of antibody size and charge variants.
Assuntos
Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Ânions/química , Cátions/química , Fracionamento Químico/métodosRESUMO
Hemoglobin (Hb) is an allosteric tetrameric protein made up of alphabeta heterodimers. The alpha and beta chains are similar, but are chemically and structurally distinct. To investigate dynamical differences between the chains, we have prepared tetramers in which the chains are isotopically distinguishable, via reconstitution with (15)N-heme. Ligand recombination and heme structural evolution, following HbCO dissociation, was monitored with chain selectivity by resonance Raman (RR) spectroscopy. For alpha but not for beta chains, the frequency of the nu(4) porphyrin breathing mode increased on the microsecond time scale. This increase is a manifestation of proximal tension in the Hb T-state, and its time course is parallel to the formation of T contacts, as determined previously by UVRR spectroscopy. Despite the localization of proximal constraint in the alpha chains, geminate recombination was found to be equally probable in the two chains, with yields of 39 +/- 2%. We discuss the possibility that this equivalence is coincidental, in the sense that it arises from the evolutionary pressure for cooperativity, or that it reflects mechanical coupling across the alphabeta interface, evidence for which has emerged from UVRR studies of site mutants.
Assuntos
Monóxido de Carbono , Hemoglobinas/química , Subunidades Proteicas/química , Análise Espectral Raman/métodos , Humanos , Cinética , Isótopos de Nitrogênio , Porfirinas , Estrutura Quaternária de Proteína , alfa-Globinas/química , Globinas beta/químicaRESUMO
Time-resolved resonance Raman (RR) spectra are reported for hemoglobin (Hb) tetramers, in which the alpha and beta chains are selectively substituted with mesoheme. The Soret absorption band shift in mesoheme relative to protoheme permits chain-selective recording of heme RR spectra. The evolution of these spectra following HbCO photolysis shows that the geminate recombination rates and the yields are the same for the two chains, consistent with recent results on (15)N-heme isotopomer hybrids. The spectra also reveal systematic shifts in the deoxyheme nu (4) and nu (Fe-His) RR bands, which are anticorrelated. These shifts are resolved for the successive intermediates in the protein structure, which have previously been determined from time-resolved UV RR spectra. Both chains show Fe-His bond compression in the immediate photoproduct, which relaxes during the formation of the first intermediate, R(deoxy) (0.07 micros), in which the proximal F-helix is proposed to move away from the heme. Subsequently, the Fe-His bond weakens, more so for the alpha chains than for the beta chains. The weakening is gradual for the beta chains, but is abrupt for the alpha chains, coinciding with completion of the R-T quaternary transition, at 20 micros. Since the transition from fast- to slow-rebinding Hb also occurs at 20 micros, the drop in the alpha chain nu (Fe-His) supports the localization of ligation restraint to tension in the Fe-His bond, at least in the alpha chains. The mechanism is more complex in the beta chains.
Assuntos
Hemoglobina A/química , Mesoporfirinas/química , Análise Espectral Raman/métodos , alfa-Globinas/química , Globinas beta/química , Monóxido de Carbono/química , Eritrócitos/química , Heme/química , Hemoglobina A/isolamento & purificação , Humanos , Fotólise , Conformação ProteicaRESUMO
The ability of ultraviolet resonance Raman (UVRR) spectroscopy to monitor a host of structurally sensitive protein vibrational modes, the amide I, II, III and S regions, makes it a potentially powerful tool for the visualization of equilibrium and non-equilibrium secondary structure changes in even the most difficult peptide samples. However, it is difficult to unambiguously resolve discrete secondary structure-derived UVRR spectral signatures independently of one another as each contributes an unknown profile to each of the spectrally congested vibrational modes. This limitation is compounded by the presence of aromatic side chains, which introduce additional overlapping vibrational modes. To address this, we have exploited an often overlooked tool for alleviating this spectral overlap by utilizing the differential excitability of the vibrational modes associated with alpha-helices and coil moieties, in the deep UV. The differences in the resonance enhancements of the various structurally associated vibrational modes yields an added dimensionality in the spectral data sets making them multi-way in nature. Through a 'chemically relevant' shape-constrained multivariate curve resolution-alternating least squares (MCR-ALS) analysis, we were able to deconvolute the complex amide regions in the multi-excitation UVRR spectrum of the protein myoglobin, giving us potentially useful 'pure' secondary structure-derived contributions to these individual vibrational profiles.
Assuntos
Motivos de Aminoácidos , Mioglobina/química , Estrutura Secundária de Proteína , Humanos , Análise dos Mínimos Quadrados , Espectrofotometria Ultravioleta/métodos , Análise Espectral Raman/métodosRESUMO
Yersinia Protein Tyrosine Phosphatase (YopH) is the most efficient enzyme among all known PTPases and relies on its catalytic loop movements for substrate binding and catalysis. Fluorescence, NMR, and UV resonance Raman (UVRR) techniques have been used to study the thermodynamic and dynamic properties of the loop motions. In this study, a computational approach based on the pathway refinement methods nudged elastic band (NEB) and harmonic Fourier beads (HFB) has been developed to provide structural interpretations for the experimentally observed kinetic processes. In this approach, the minimum potential energy pathways for the loop open/closure conformational changes were determined by NEB using a one-dimensional global coordinate. Two dimensional data analyses of the NEB results were performed as an efficient method to qualitatively evaluate the energetics of transitions along several specific physical coordinates. The free energy barriers for these transitions were then determined more precisely using the HFB method. Kinetic parameters were estimated from the energy barriers using transition state theory and compared against experimentally determined kinetic parameters. When the calculated energy barriers are calibrated by a simple "scaling factor", as have been done in our previous vibrational frequency calculations to explain the ligand frequency shift upon its binding to protein, it is possible to make structural interpretations of several observed enzyme dynamic rates. For example, the nanosecond kinetics observed by fluorescence anisotropy may be assigned to the translational motion of the catalytic loop and microsecond kinetics observed in fluorescence T-jump can be assigned to the loop backbone dihedral angle flipping. Furthermore, we can predict that a Trp354 conformational conversion associated with the loop movements would occur on the tens of nanoseconds time scale, to be verified by future UVRR T-jump studies.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Tirosina Fosfatases/metabolismo , Yersinia/enzimologia , Biocatálise , Conformação Proteica , Proteínas Tirosina Fosfatases/química , TermodinâmicaRESUMO
Tryptophan (Trp) oxidation in proteins leads to a number of events, including changes in color, higher order structure (HOS), and biological activity. We describe here a number of new findings through a comprehensive characterization of 6 monoclonal antibodies (mAbs) following selective oxidation of Trp residues by 2,2'-azobis(2-amidinopropane) dihydrochloride. Fluorescence spectroscopy, in combination with second derivative analysis, demonstrates that the loss of Trp fluorescence intensity is a sensitive indicator of Trp oxidation in mAbs. Size-exclusion chromatography with UV and intrinsic Trp fluorescence detection was demonstrated to be a useful method to monitor Trp oxidation levels in mAbs. Furthermore, the Trp oxidation levels measured by size-exclusion chromatography with UV and intrinsic Trp fluorescence detection were found to be in agreement with the values obtained from tryptic peptide mapping by liquid chromatography with mass spectrometric detection and correlate with the total solvent accessible surface area of the exposed Trp residues from in silico modeling. Finally, near-UV circular dichroism and Raman spectroscopy were used to evaluate the impact of Trp oxidation on HOS and identify specific oxidation products, respectively. This work demonstrates that protein HOS is altered on Trp oxidation in mAbs and multiple spectroscopic markers can be used to monitor the molecule-dependent Trp oxidation behavior.