ABSTRACT
A major limitation of intact protein fragmentation is the lack of sequence coverage within proteins' interiors. We show that collisionally activated dissociation (CAD) produces extensive internal fragmentation within proteins' interiors that fill the existing gaps in sequence coverage, including disulfide loop regions that cannot be characterized using terminal fragments. A barrier to the adoption of internal fragments is the lack of methods for their generation and assignment. To provide these, we explore the effects of protein size, mass accuracy, internal fragment size, CAD activation energy, and data preprocessing upon the production and identification of internal fragments. We also identify and mitigate the major source of ambiguity in internal fragment identification, which we term "frameshift ambiguity." Such ambiguity results from sequences containing any "middle" portion surrounded by the same composition on both termini, which upon fragmentation can produce two internal fragments of identical mass, yet out of frame by one or more amino acids (e.g., TRAIT producing TRAI or RAIT). We show that such instances permit the a priori assignment of the middle sequence portion. This insight and our optimized methods permit the unambiguous assignment of greater than 97% of internal fragments using only the accurate mass. We show that any remaining ambiguity in internal fragment assignment can be removed by consideration of fragmentation propensities or by (pseudo)-MS3. Applying these methods resulted in a 10-fold and 43-fold expanded number of identified ions, and a concomitant 7- and 16-fold increase in fragmentation sites, respectively, for native and reduced forms of a disease-associated SOD1 variant.
ABSTRACT
The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.
Subject(s)
Antibodies, Monoclonal , Mass Spectrometry/methods , Proteomics/methods , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Complementarity Determining Regions/analysis , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Humans , MiceABSTRACT
The unfolded protein response (UPR) senses defects in the endoplasmic reticulum (ER) and orchestrates a complex program of adaptive cellular remodeling. Increasing evidence suggests an important relationship between lipid homeostasis and the UPR. Defects in the ER membrane induce the UPR, and the UPR in turn controls the expression of some lipid metabolic genes. Among lipid species, the very-long-chain fatty acids (VLCFAs) are relatively rare and poorly understood. Here, we show that loss of the VLCFA-coenzyme A synthetase Fat1, which is essential for VLCFA utilization, results in ER stress with compensatory UPR induction. Comprehensive lipidomic analyses revealed a dramatic increase in membrane saturation in the fat1Δ mutant, likely accounting for UPR induction. In principle, this increased membrane saturation could reflect adaptive membrane remodeling or an adverse effect of VLCFA dysfunction. We provide evidence supporting the latter, as the fat1Δ mutant showed defects in the function of Ole1, the sole fatty acyl desaturase in yeast. These results indicate that VLCFAs play essential roles in protein quality control and membrane homeostasis and suggest an unexpected requirement for VLCFAs in Ole1 function.
Subject(s)
Endoplasmic Reticulum Stress/physiology , Endoplasmic Reticulum/metabolism , Unfolded Protein Response/physiology , Coenzyme A Ligases/metabolism , Endoplasmic Reticulum/physiology , Fatty Acid Transport Proteins/metabolism , Fatty Acids/metabolism , Homeostasis , Lipid Metabolism/physiology , Lipids/physiology , Membranes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Unfolded Protein Response/geneticsABSTRACT
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) provides a unique in situ chemical profile that can include drugs, nucleic acids, metabolites, lipids, and proteins. MSI of individual cells (of a known cell type) affords a unique insight into normal and disease-related processes and is a prerequisite for combining the results of MSI and other single-cell modalities (e.g. mass cytometry and next-generation sequencing). Technological barriers have prevented the high-throughput assignment of MSI spectra from solid tissue preparations to their cell type. These barriers include obtaining a suitable cell-identifying image (e.g. immunohistochemistry) and obtaining sufficiently accurate registration of the cell-identifying and MALDI-MS images. This study introduces a technique that overcame these barriers by assigning cell type directly from mass spectra. We hypothesized that, in MSI from mice with a defined fluorescent protein expression pattern, the fluorescent protein's molecular ion could be used to identify cell cohorts. A method was developed for the purification of enhanced yellow fluorescent protein (EYFP) from mice. To determine EYFP's molecular mass for MSI studies, we performed intact mass analysis and characterized the protein's primary structure and post-translational modifications through various techniques. MALDI-MSI methods were developed to enhance the detection of EYFP in situ, and by extraction of EYFP's molecular ion from MALDI-MS images, automated, whole-image assignment of cell cohorts was achieved. This method was validated using a well-characterized mouse line that expresses EYFP in motor and sensory neurons and should be applicable to hundreds of commercially available mice (and other animal) strains comprising a multitude of cell-specific fluorescent labels.
Subject(s)
Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Imaging/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Animals , Mice , Molecular Weight , Neurons/metabolismABSTRACT
Conventional metabolomic methods include extensive sample preparation steps and long analytical run times, increasing the likelihood of processing artifacts and limiting high throughput applications. We present here in vitro liquid extraction surface analysis mass spectrometry (ivLESA-MS), a variation on LESA-MS, performed directly on adherent cells grown in 96-well cell culture plates. To accomplish this, culture medium was aspirated immediately prior to analysis, and metabolites were extracted using LESA from the cell monolayer surface, followed by nano-electrospray ionization and MS analysis in negative ion mode. We applied this platform to characterize and compare lipidomic profiles of multiple breast cancer cell lines growing in culture (MCF-7, ZR-75-1, MDA-MB-453, and MDA-MB-231) and revealed distinct and reproducible lipidomic signatures between the cell lines. Additionally, we demonstrated time-dependent processing artifacts, underscoring the importance of immediate analysis. ivLESA-MS represents a rapid in vitro metabolomic method, which precludes the need for quenching, cell harvesting, sample preparation, and chromatography, significantly shortening preparation and analysis time while minimizing processing artifacts. This method could be further adapted to test drugs in vitro in a high throughput manner.
Subject(s)
Lipids/analysis , Spectrometry, Mass, Electrospray Ionization/methods , Cell Line, Tumor , Chromatography, High Pressure Liquid , Culture Media/chemistry , Humans , Lipids/isolation & purification , Liquid-Liquid Extraction , Metabolomics/methods , NanotechnologyABSTRACT
Stable isotope labeling techniques for quantitative top-down proteomics face unique challenges. These include unpredictable mass shifts following isotope labeling, which impedes analysis of unknown proteins and complex mixtures and exponentially greater susceptibility to incomplete isotope incorporation, manifesting as broadening of labeled intact protein peaks. Like popular bottom-up isotope labeling techniques, most top-down labeling methods are restricted to defined media/feed as well as amino acid auxotrophic organisms. We present a labeling method optimized for top-down proteomics that overcomes these challenges. We demonstrated this method through the spiking of 13C-sugar or 2H-water into standard laboratory feedstocks, resulting in tunable intact protein mass increases (TIPMI). After mixing of labeled and unlabeled samples, direct comparison of light and heavy peaks allowed for the relative quantitation of intact proteins in three popular model organisms, including prokaryotic and eukaryotic microorganisms and an animal. This internal standard method proved to be more accurate than label-free quantitation in our hands. Advantages over top-down SILAC include working equally well in nutrient-rich media, conceivably expanding applicability to any organism and all classes of biomolecules, not requiring high-resolving power MS for quantitation and being relatively inexpensive.