Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies 相似文献
Genomic approaches to characterizing bacterial communities are revealing significant differences in diversity and composition between environments. But bacterial distributions have not been mapped at a global scale. Although current community surveys are way too sparse to map global diversity patterns directly, there is now sufficient data to fit accurate models of how bacterial distributions vary across different environments and to make global scale maps from these models. We apply this approach to map the global distributions of bacteria in marine surface waters. Our spatially and temporally explicit predictions suggest that bacterial diversity peaks in temperate latitudes across the world''s oceans. These global peaks are seasonal, occurring 6 months apart in the two hemispheres, in the boreal and austral winters. This pattern is quite different from the tropical, seasonally consistent diversity patterns observed for most macroorganisms. However, like other marine organisms, surface water bacteria are particularly diverse in regions of high human environmental impacts on the oceans. Our maps provide the first picture of bacterial distributions at a global scale and suggest important differences between the diversity patterns of bacteria compared with other organisms. 相似文献
The objective of the present study was to isolate and purify the protein fraction(s) of llama seminal plasma responsible for
the ovulation-inducing effect of the ejaculate. 相似文献
Rapid, sensitive, and specific virus detection is an important component of clinical diagnostics. Massively parallel sequencing enables new diagnostic opportunities that complement traditional serological and PCR based techniques. While massively parallel sequencing promises the benefits of being more comprehensive and less biased than traditional approaches, it presents new analytical challenges, especially with respect to detection of pathogen sequences in metagenomic contexts. To a first approximation, the initial detection of viruses can be achieved simply through alignment of sequence reads or assembled contigs to a reference database of pathogen genomes with tools such as BLAST. However, recognition of highly divergent viral sequences is problematic, and may be further complicated by the inherently high mutation rates of some viral types, especially RNA viruses. In these cases, increased sensitivity may be achieved by leveraging position-specific information during the alignment process. Here, we constructed HMMER3-compatible profile hidden Markov models (profile HMMs) from all the virally annotated proteins in RefSeq in an automated fashion using a custom-built bioinformatic pipeline. We then tested the ability of these viral profile HMMs (“vFams”) to accurately classify sequences as viral or non-viral. Cross-validation experiments with full-length gene sequences showed that the vFams were able to recall 91% of left-out viral test sequences without erroneously classifying any non-viral sequences into viral protein clusters. Thorough reanalysis of previously published metagenomic datasets with a set of the best-performing vFams showed that they were more sensitive than BLAST for detecting sequences originating from more distant relatives of known viruses. To facilitate the use of the vFams for rapid detection of remote viral homologs in metagenomic data, we provide two sets of vFams, comprising more than 4,000 vFams each, in the HMMER3 format. We also provide the software necessary to build custom profile HMMs or update the vFams as more viruses are discovered (http://derisilab.ucsf.edu/software/vFam). 相似文献
With the increasing availability of microbiome 16S data, network estimation has become a useful approach to studying the interactions between microbial taxa. Network estimation on a set of variables is frequently explored using graphical models, in which the relationship between two variables is modeled via their conditional dependency given the other variables. Various methods for sparse inverse covariance estimation have been proposed to estimate graphical models in the high-dimensional setting, including graphical lasso. However, current methods do not address the compositional count nature of microbiome data, where abundances of microbial taxa are not directly measured, but are reflected by the observed counts in an error-prone manner. Adding to the challenge is that the sum of the counts within each sample, termed “sequencing depth,” is an experimental technicality that carries no biological information but can vary drastically across samples. To address these issues, we develop a new approach to network estimation, called BC-GLASSO (bias-corrected graphical lasso), which models the microbiome data using a logistic normal multinomial distribution with the sequencing depths explicitly incorporated, corrects the bias of the naive empirical covariance estimator arising from the heterogeneity in sequencing depths, and builds the inverse covariance estimator via graphical lasso. We demonstrate the advantage of BC-GLASSO over current approaches to microbial interaction network estimation under a variety of simulation scenarios. We also illustrate the efficacy of our method in an application to a human microbiome data set.
The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be 'Gram-positive,' whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be 'Gram-negative.' This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as Gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool. 相似文献