Development of a dozen or more gentle ionization methods—for example, MALDI (matrix-assisted laser desorption/ionization) and electrospray ionization— have made mass spectrometric peptide and protein analysis routine.
“When I started in this business twenty-five years ago, we couldn’t analyze insulin, a peptide with a [molecular weight] of 5,808 dalton [Da],” says Steven Fischer, director of marketing for academia and government at Agilent Technologies (Santa Clara, CA).
Aided by computerization, miniaturization, and highspeed electronics, advances in protein MS came “fast and furious,” Fischer adds. Today’s “expert” upper mass limit is 1.4 million Da, with routine analysis hitting about 145 kDa.
Some labs perform intact protein analysis, but that approach has become rare. Not much information is accessible beyond the gross molecular weight. Obtaining the most information from a peptide or a protein requires subjecting the sample to proteolytic analysis followed by MS/MS to discern fine structural details. This approach, bottom-up proteomics, depends heavily on sample preparation.
Through its AssayMAP Bravo Platform liquid-handling workstation, Agilent offers a fully automated system for digesting proteins and preparing them for further analysis. Automation and consistency are major issues in quality assessments of therapeutic proteins. “Poor digestion leads to much more complicated data,” Fischer explains.
Top, bottom, middle
Top-down MS protein analysis begins with the intact protein, whereas bottom-up analysis begins with enzymatically generated fragments. Recently, middle-down analysis has been gaining adherents as well. Here, midsized proteins are fragmented using electron transfer dissociation and subsequently sequence-annotated. Electron transfer dissociation can be used with or without additional ion mobility separation. “The advantages of the technique is it preserves posttranslational modifications—PTMs,” says Hans Vissers, senior manager of science operations, health sciences research at Waters (Milford, MA). “Challenges include low sensitivity and software availability.”
Sample preparation is critical for this method but is becoming more routine. Automation of digestion protocols aids in providing assay robustness, repeatability, and standardization. Enrichment for the analysis of glycopeptides and phosphopeptides addresses concentration dynamic ranges within samples, but universal protocols are not yet available, according to Vissers. “Extraction protocols for membrane proteins and the affinity selection of low-abundance plasma proteins for discovery and validation studies are quite specialized.”
Trends in hardware and software include the development of combined qualitative and quantitative data-independent techniques, along with informatics, with or without the use of compound libraries. “Ion mobility-assisted data-independent acquisition and scanning quadrupole DIA [data-independent analysis] methods are becoming mainstream, with the data analyzed using both commercial and open source informatics. These techniques are equally suited for metabolic profiling, an emerging discipline in omics fields,” Vissers says.
“Bottom-up protein MS allows us to identify the amino acid sequence of the protein, as well as any posttranslational modifications that may be present,” says Kelli Jonakin, PhD, senior scientist and global marketing manager at SCIEX (Framingham, MA). In addition to identifying PTMs such as oxidation, deamidation, and important glycosylations, these MS analyses can also identify sequence variants in the protein, all of which can affect a drug’s safety, efficacy, and pharmacokinetics.
Bottom-up high-resolution MS can identify amino acids from individual peptides, along with many PTMs of interest. It resolves not just the sequence (or variances), but also the exact location of glycosylation sites and, through MS/MS, analysis of the glycans themselves.
When glycosylation composition and patterns are of interest, capillary electrophoresis electrospray ionization (CESI) is the separation of choice, as it can maximize ionization efficiency and minimize ion suppression. “This is a great benefit for hydrophilic species such as glycopeptides, which can be challenging to analyze by traditional liquid chromatography separations,” Jonakin explains.
CESI coupled with high-resolution MS can provide 100 percent sequence coverage, plus identification of all relevant PTMs and glycosylations. MS fragmentation of glycanbearing peptides results in a highly complex spectrum of peptide and glycan fragmentation products. With an appropriate mass analyzer— for example, the SCIEX TripleTOF™ 6600 MS with CESI interface—it’s possible to acquire all levels of data simultaneously. “What used to take three or four experiments for sequence identification, PTMs, and glycan analysis, is now achievable [with] a single injection,” Jonakin says.
All hardware vendors sell software for basic control and data analysis. The programs are analysis modules in themselves. As research groups expand into advanced applications, the algorithms and capabilities of “native” software fall short.
Thus the robust industry of third-party MS analysis software.
Protein Metrics (San Carlos, CA) offers a suite of specialized proteomics packages—for peptide mapping, intact protein mass determination, and posttranslational modification analysis, for example—in addition to a product for proteomic liquid chromatography.
“Protein characterization is no longer simply a matter of identifying molecules but of learning everything you can about them as early as possible,” says St. John Skilton, PhD, senior director of marketing and sales at Protein Metrics. “Third-party software fills significant gaps in MS software left behind by the continued pace and demanding requirements of modern protein analysis.”
Biopharmaceuticals are highly diverse, even within one batch of one product. Posttranslational modifications such as sequence variance, glycosylation, deamidation, oxidation, and others—and the relative occurrences of these changes—are considered key quality attributes. Drug developers are eager for analysis methods that uncover these characteristics, but hardware vendors generally lack the resources or focus to develop such tools. That’s where third-party developers like Protein Metrics enter the picture.
A good deal of the MS proteomics work these days focuses on biosimilars—the biotherapeutics equivalent of generic drugs. Biosimilar developers must demonstrate comparability with the originator project in terms of efficacy and, equally importantly, chemical structure. MS techniques have become the go-to methods for assessing biosimilarity, but as Skilton notes, the road from raw spectral data to structural understanding is not straightforward.
“MS by itself doesn’t tell you if proteins are similar; it only generates raw data for you to play with. Additionally, analysts are often looking for very minute details, at very low levels in many cases. For a human to troll through that data is well-nigh impossible.”
Jonakin, whose company collaborates with Protein Metrics, calls its software “fantastic,” but notes that her own company’s BiopharmaView™ software is also highly specialized for these complex biotherapeutic analyses.
The user experience—having an easy-tounderstand readout that uncovers sequence and PTMs—is a significant part of MS data processing. “BiopharmaView software provides that simplicity but without losing the critical tie-back to the raw data. Becoming too reliant on a table or graph that the software creates for you is risky, especially when the analyst can’t go back in and see the raw data to troubleshoot it when problems arise,” she says.
Are you prepping correctly?
Proper sample preparation for mass spectrometrybased analysis is essential to obtain reproducible and high-quality results. Find out some of the latest trends in MS sample prep.
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