When employing mass spectrometry to characterize structural variants or modifications in proteins, measuring the mass of the intact molecule offers a broad overview of the entire protein but lacks the ability to pinpoint the precise locations of these variations or modifications. Essentially, intact mass measurement provides no structural resolution. To achieve comprehensive structural insights, it becomes imperative to cleave the protein into smaller fragments before subjecting it to mass analysis. This cleavage process can occur either in solution or in the gas phase. When fragments are predominantly generated in solution, each fragment is individually analyzed by the mass spectrometer, and the molecule is reconstructed based on this information. This method is known as the "up" type approach. Conversely, when fragments are primarily generated in the gas phase within the mass spectrometer, the instrument directly analyzes the entire molecule, which is termed the "down" type method.
In the realm of glycoprotein characterization using mass spectrometry, three primary strategies are commonly employed: top-down, middle-up, and bottom-up (with a focus on peptide and glycan fragment ions). Among these, the bottom-up approach reigns supreme for structural characterization due to its capability to analyze both the protein sequence and the glycosylation sites on amino acid residues. This approach involves enzymatic digestion of the protein into peptides, followed by mass spectrometric analysis of the resulting peptide fragments. By scrutinizing the peptide sequences and identifying glycosylation sites, bottom-up methodology provides comprehensive insights into the structural intricacies of glycoproteins.
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Bottom-up Structural Characterization
The most frequently used technique for confirming protein sequences or characterizing modifications at the residue level is the bottom-up approach. In bottom-up proteomics, proteins are digested into peptides using proteases, followed by LC/MS or more commonly LC/MS/MS analysis. The combination of proteolytic digestion and LC/MS/MS offers high structural resolution but is typically the most material- and time-consuming, as well as labor-intensive in mass spectrometry analysis. Care must be taken during digestion to prevent artifacts such as the loss of labile modifications like succinimide and amino acid rearrangements, which can lead to incorrect conclusions. Bottom-up is widely used to confirm protein sequences and to characterize post-translational, processing, and storage modifications, as well as to map disulfide bonds.
Proteolysis
Typically, monoclonal antibodies (mAbs) are reduced and alkylated under denaturing conditions before proteolysis using trypsin or Lys-C. Less commonly used proteases include Glu-C (V8) and Asp-N, and occasionally, chemical methods such as cyanogen bromide (CNBr) cleavage are employed.
Most methods cut the protein into peptides of suitable sizes for effective fragmentation in MS/MS experiments. It is noteworthy that on most commercial instruments, large peptides (>4 kDa) are challenging to characterize through MS/MS, and very small peptides (2-3 residues) often get lost due to poor retention on reverse-phase columns.
LC/MS and LC/MS/MS
Initially, MALDI-TOF instruments were widely used for determining the mass of peptides produced from the proteolysis of mAbs and other proteins for structural confirmation and analysis of isoforms/modifications. MALDI-TOF offers ease of use and requires less consumables and time compared to ESI-MS. However, due to the convenience of LC-tandem and the superior quality of tandem mass spectrometry required for peptide identification, most bottom-up experiments on mAbs are now conducted on ESI instruments.
In LC/MS/MS peptide mapping experiments, the challenge often lies in the proteolysis of the mAb. A significant amount of protein material is typically required for successful digestion. Once complete digestion is achieved, the sensitivity of MS detection, thanks to advances in modern instrumentation, is usually not a problem. Most LC/MS/MS peptide mapping experiments are performed on narrow bore RP-UPLC columns to achieve optimal chromatographic resolution.
Disulfide Bond Determination
To determine the disulfide bond connectivity within a protein, digestion must be carried out while disulfide bonds remain intact. Under normal conditions, monoclonal antibody molecules are very stable and difficult to digest, making the characterization of disulfide bonds within them challenging. Key to successful digestion is denaturing the mAb under harsh conditions, such as with 6M guanidine HCl at high temperatures. During denaturation, it is crucial to have alkylation agents like iodoacetate (IAA), iodoacetamide (IAM), or N-ethylmaleimide (NEM) present to prevent disulfide scrambling by capping all free thiols. NEM is often preferred due to its activity at lower pHs. Before digestion, denaturants must be diluted to preserve protease activity. Digestion typically uses trypsin or Lys-C. Subsequently, a portion of the sample is further reduced with DTT or TCEP for LC/MS/MS peptide mapping comparison with unreduced maps to determine disulfide connectivity. Disulfide bonds near the hinge region are often detected using Edman sequencing or partial reduction under mild acidic conditions in the presence of NEM or M-biotin.
Another method for determining disulfide bonds in mAbs is analyzing the non-reduced digest using LC/MS in negative ion mode. In this mode, disulfide bonds are effectively cleaved, revealing the linked chains, akin to gas-phase reduction of disulfide bonds.
Schematic workflow of the hybrid MS approaches for the structural analysis of protein heterogeneity (Yang et al., 2016).
Middle-Up Structural Characterization
Determining the mass of intact monoclonal antibodies (mAbs) provides an essential, yet basic, overview of the protein's molecular weight but does not delve into the finer details of its structural intricacies. To uncover these details, breaking down the protein into smaller segments is necessary before conducting mass spectrometric analysis. The "middle-up" approach offers a sophisticated alternative by sectioning mAbs into several larger, yet manageable, fragments that still retain significant structural information.
One practical technique within the middle-up strategy is the reduction of disulfide bonds to separate the protein into its constituent heavy and light chains. This segmentation allows for individual mass analysis of each chain, enabling precise confirmation of each chain's structure. It also helps in pinpointing variations and modifications that might occur within each chain, which are crucial for understanding the functionality and behavior of mAbs in biological systems.
The complete reduction of all disulfide bonds is typically performed under denaturing conditions, which ensures that all bonding interactions are sufficiently disrupted to allow for a thorough analysis. However, in scenarios where preserving the natural state of the protein as much as possible is crucial, interchain disulfide bonds can be selectively targeted for reduction while keeping intrachain bonds intact. This selective reduction not only aids in maintaining some aspects of the protein's original conformation but is also instrumental in analyzing and confirming the structural integrity and modifications of mAbs, such as glycosylation patterns on the heavy chains, which are vital for their biological efficacy.
Furthermore, another prevalent method in middle-up approaches is the use of proteolytic enzymes such as papain or pepsin under native conditions to cleave the mAbs. This enzymatic treatment is usually focused around the hinge regions of many IgG type molecules, effectively producing fragments like Fab, F(ab)2, and Fc. This method is particularly beneficial because it mimics the natural processing of antibodies and helps preserve important functional domains. However, it is noteworthy that IgG2 molecules show resistance to enzymatic digestion under these conditions, presenting a challenge that requires careful methodological adjustments.
The enzymatically derived fragments, once generated, can undergo further reduction to break them down into smaller pieces, enhancing the detail available in mass spectrometric analysis. This step is critical as it provides a deeper look into the subtle structural nuances compared to analyzing the full mAb mass, hence offering a richer understanding of the antibody's structure and function.
By employing these techniques, the middle-up approach not only preserves a considerable amount of the structural and functional integrity of the antibodies but also allows for detailed characterization. This method effectively bridges the gap between the need for detailed structural information and maintaining the functional aspects of the antibodies, making it an invaluable strategy in the structural characterization of monoclonal antibodies.
Top-Down Structural Characterization
Although bottom-up methods indeed offer a wealth of structural information, their implementation often entails laborious procedures and potential challenges. These challenges include the need for significant sample quantities, the risk of introducing artifacts during digestion, and time-consuming sample preparation protocols. For samples with limited quantities or low concentrations, such as trace components obtained from chromatographic separations, conducting bottom-up analyses necessitates multiple rounds of collection and concentration to generate sufficient material for successful enzymatic digestion. In such scenarios, researchers often turn to alternative approaches like top-down and middle-down methodologies, which offer rapid and convenient solutions for obtaining valuable sequence information.
Top-down mass spectrometry stands out as a powerful technique wherein intact protein molecules are introduced directly into the mass spectrometer without undergoing enzymatic or chemical degradation in solution. Structural information is then gleaned by analyzing the fragmentation patterns of the intact molecule within the mass spectrometer. This method is particularly efficient for rapidly characterizing small to medium-sized proteins. However, due to the substantial number of highly charged fragment ions generated during fragmentation, high-resolution mass spectrometers are typically employed for top-down analysis.
For instance, a Q-TOF instrument with a resolution of 10,000 is capable of providing significant structural insights for proteins up to 10,000 Da in mass. Nonetheless, for larger proteins, instruments with even higher resolution capabilities, such as Fourier Transform Ion Cyclotron Resonance (FT-ICR MS) or Orbitrap, are preferred. These instruments enable more precise and detailed analysis, making them well-suited for characterizing larger proteins and elucidating their complex structural features with greater accuracy.
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Middle-Down Structural Characterization
Middle-down structural characterization emerges as a significant alternative to top-down methodologies, particularly in addressing the structural resolution limitations inherent in top-down approaches. The middle-down strategy begins by segmenting proteins into several larger fragments, serving as a crucial intermediary step before detailed analysis. These larger fragments are obtained through various means, including the reduction of disulfide bonds or limited proteolytic digestion.
One common method in middle-down approaches involves the strategic reduction of disulfide bonds, a chemical reaction that facilitates the separation of proteins into manageable fragments. Unlike enzymatic reactions, which may have stringent concentration requirements and can involve expensive enzymes, chemical reactions offer flexibility in reagent usage. This flexibility allows for the use of ample chemical reagents without the constraints of enzyme concentration, making middle-down approaches particularly advantageous for diluted or small-volume samples.
Following the segmentation process, these fragments, enriched with structural information, are introduced into a mass spectrometer for MS/MS analysis. This analytical technique enables the elucidation of fragment structures and provides insights into the overall protein structure. Compared to top-down methodologies, middle-down approaches offer the potential to enhance structural resolution, thanks to the larger fragment sizes and the elimination of certain limitations associated with enzymatic reactions.
Moreover, middle-down experiments offer an effective means of achieving high structural resolution without the logistical challenges of enzyme concentration and cost. This aspect is particularly advantageous for researchers working with limited sample volumes or facing budget constraints. By leveraging chemical reactions and larger fragment sizes, middle-down structural characterization provides a robust and cost-effective approach to unraveling complex protein structures and gaining deeper insights into their functionalities.
Reference
- Yang, Yang , et al. "Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity." Nature Communications 7(2016):13397.