Microsequence analysis of peptides and proteins. IX. Manual gas-phase microsequencing of multiple samples

A novel apparatus for performing manual gas-phase Edman chemistry on protein and peptide samples is described. Edman chemistry is performed in 6 to 10 Teflon continuous flow reactors (CFR), previously described by J.E. Shively et al. (1987) Anal. Biochem. 163, 517-529). The CFRs are packed with 10-15 mg of Polybrene-coated spherical silica (Porasil B, Waters Associates). The gas-phase coupling reagent and cleavage reagent are 5% aqueous triethylamine and anhydrous trifluoroacetic acid, respectively, delivered by a stream of argon gas. The delivery of the gas-phase reagents is manually controlled with Hamilton 3-way valves and 2-way valves, and that of the solvents, ethyl acetate and butyl chloride, by syringe pipetting. The average cycle time is 15-20 min for 6 to 10 samples run simultaneously. Conversion of the anilinothiazolinone to phenylthiohydantoin (PTH) amino acid derivatives is accomplished manually with 25% aqueous trifluoroacetic acid. The PTH amino acids are analyzed by reversed-phase HPLC using an autosampler for handling multiple samples. Excellent results were obtained in the 100-200 pmol range. Protein samples can be sequenced from 15-20 cycles, and peptide samples usually to the COOH terminus. Initial yields ranged from 30 to 60% and repetitive yields ranged from 90 to 96%. The sample washout and size of background peaks are significantly reduced, compared to older methods of manual sequence analysis. The yields and background signal to noise are comparable to automated gas-phase Edman chemistry. The improved manual Edman described represents a low cost alternative to automated sequence analysis, and has the advantage being able to process multiple samples simultaneously.     

Nanostructure-initiator mass spectrometry: a protocol for preparing and applying NIMS surfaces for high-sensitivity mass analysis

Nanostructure-initiator mass spectrometry (NIMS) is a new surface-based MS technique that uses a nanostructured surface to trap liquid ('initiator') compounds. Analyte materials adsorbed onto this 'clathrate' surface are subsequently released by laser irradiation for mass analysis. In this protocol, we describe the preparation of NIMS surfaces capable of producing low background and high-sensitivity mass spectrometric measurement using the initiator compound BisF17. Examples of analytes that adsorb to this surface are small molecules, drugs, lipids, carbohydrates and peptides. Typically, NIMS is used to analyze samples ranging from simple analytical standards and proteolytic digests to more complex samples such as tissues, cells and biofluids. Critical experimental considerations of NIMS are described. Specifically, NIMS sensitivity is examined as a function of pre-etch cleaning treatment, etching current density, etching time, initiator composition, sample concentration, sample deposition method and laser fluence. Typically, NIMS surface preparation can be completed in less than 2 h. Subsequent sample preparation requires 1-5 min, depending on sample deposition method. Mass spectrometric data acquisition typically takes 1-30 s per sample.     

Deep learning image analysis models pretrained on daily objects are useful for the preliminary characterization of particulate pharmaceutical samples

Visible and subvisible particles are a quality attribute in sterile pharmaceutical samples. A common method for characterizing and quantifying pharmaceutical samples containing particulates is imaging many individual particles using high-throughput instrumentation and analyzing the populations data. The analysis includes conventional metrics such as the particle size distribution but can be more sophisticated by interpreting other visual/morphological features. To avoid the hurdles of building new image analysis models capable of extracting such relevant features from scratch, we propose using well-established pretrained deep learning image analysis models such as EfficientNet. We demonstrate that such models are useful as a prescreening tool for high-level characterization of biopharmaceutical particle image data. We show that although these models are originally trained for completely different tasks (such as the classification of daily objects in the ImageNet database), the visual feature vectors extracted by such models can be useful for studying different types of subvisible particles. This applicability is illustrated through multiple case studies: (i) particle risk assessment in prefilled syringe formulations containing different particle types such as silicone oil, (ii) method comparability with the example of accelerated forced degradation, and (iii) excipient influence on particle morphology with the example of Polysorbate 80 (PS80). As examples of agnostic applicability of pretrained models, we also elucidate the application to two high-throughput microscopy methods: microflow and background membrane imaging. We show that different particle populations with different morphological and visual features can be identified in different samples by leveraging out-of-the-box pretrained models to analyze images from each sample.     

标记辅助回交育种中所需最小样本容量的近似估计

回交育种是把有利基因从供体亲本向受体亲本转移的一种有效方法,标记辅助选择可加速其进程。为了制定合理的标记辅助选择计划,育种家必须知道所需的后代群体大小。该文提出了一种估算在标记辅助回交育种中同时进行前景选择和背景选择所需群体大小的方法。在假定所需转移的目标基因座与遗传背景之间为相互独立的简化假设下,可以通过将解析方法（针对前景选择）与基于回交亲本图示基因型的模拟方法（针对背景选择）相结合,近似地估计出在每一世代中选到所需基因型的概率,进而估算出在一定概率水平下至少获得一个符合要求的个体所需的最小样本容量,用假想的例子演示了该方法的使用情况。该方法可以很方便地应用于实际的回交育种。     

Elemental imaging by EELS and EDXS in the analytical electron microscope

Electron energy loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDXS) can be used to obtain elemental maps from thin biological samples in the analytical electron microscope. The EELS is particularly sensitive for the low-atomic-number elements, including C, N, and O, as well as other elements with favorable ionization cross-sections, such as Fe. The EDXS is useful for a complementary range of atoms, such as P, S, K, and Ca. A system is described for obtaining elemental distributions in an analytical electron microscope operated in the scanning transmission mode at 100–200 keV beam energy. The spatial resolution is typically limited to 10–20 nm when a conventional source is used. A satellite microcomputer controls acquisition of EELS and EDXS data from successive pixels in an image. These data are processed “on-the-fly” by a host computer to remove the noncharacteristic background intensity. Resulting images are stored on disk and can be analyzed by means of an image display system controlled by interactive software. The technique is demonstrated with elemental maps from two samples: alveolar macrophages containing respirable particles; and pancreatic beta cells that secrete insulin.     

LC–ESI-Q-TOF-MS for faster and accurate determination of microcystins and nodularins in serum

Microcystins (MC) and nodularins (Nod) are cyclic peptide hepatotoxins and tumour promoters produced by cyanobacteria. This study deals with liquid chromatography–mass spectrometry (LC–MS) analyses of 9 major cyanobacterial peptide toxins, starting with a comparison of six small particle size reversed-phase HPLC columns, from which one, Phenomenex Synergi Hydro-RP, was chosen for further chromatography with accurate mass MS studies in a complex biological fluid, serum. The instrumentation used for the serum sample analysis included a Bruker micrO-TOF-Q-MS coupled to an Agilent 1200RR LC system. Total analysis run time per sample was 8.5 min. The Q-TOF-MS instrument was operated on auto MS–MS mode to obtain fragment ions (such as the characteristic fragment m/z 135 from Adda amino acid residue) for toxin identification purposes. Detected mass errors in serum samples were in the range of from 0.3 mDa to 9.1 mDa. The narrow mass window (±20 mDa) for mass chromatograms used in quantitation gave benefits by background noise reduction. We conclude that a LC–ESI-Q-TOF-MS instrumentation is a powerful tool for identification and quantitation of cyanobacterial peptide toxins in a biological matrix.     

Heterogeneity ratio: a measure of beta-diversity and its use in community classification

Fifteen previously proposed similarity indices are examined for the effects of sample size and/or group size (the number of samples included in a cluster). The three indices ofCλ,NESS, andC′λ are free from effects, but the former two are unsuitable for arithmetic averaging unless all of the sample sizes are equal. Thus clustering usingC′λ is found to be superior to the combination of any other similarity index and the group-average strategy. Unfortunately none of these measures have the desirable property of measuring the difference in component species among samples independent of the alpha-diversity. A new index of similarity (HR) is developed based on the assumption that community from which samples are taken is described by a logseries distribution. This new index measures the beta-diversity among samples without the influence of sample size and group size, and has the advantage that the significance of fusing samples can statistically be tested. An example clustering withHR is shown and compared with those obtained by other clustering strategies.     

Estimation of population growth or decline in genetically monitored populations

This article introduces a new general method for genealogical inference that samples independent genealogical histories using importance sampling (IS) and then samples other parameters with Markov chain Monte Carlo (MCMC). It is then possible to more easily utilize the advantages of importance sampling in a fully Bayesian framework. The method is applied to the problem of estimating recent changes in effective population size from temporally spaced gene frequency data. The method gives the posterior distribution of effective population size at the time of the oldest sample and at the time of the most recent sample, assuming a model of exponential growth or decline during the interval. The effect of changes in number of alleles, number of loci, and sample size on the accuracy of the method is described using test simulations, and it is concluded that these have an approximately equivalent effect. The method is used on three example data sets and problems in interpreting the posterior densities are highlighted and discussed.     

Components and results of a new preparation technique for automated analysis of cervical samples

Components and evaluations of a new preparative procedure for automated high-resolution analysis of cervical samples are presented. This procedure is based on sedimentation velocity separation of samples with subsequent fractionation of the separation column and centrifugal deposition of suspended cells on coated glass slides. A system for specimen collection and mailing of suspended samples is described. A new type of glass slide designed for automated analysis is presented, and centrifugal buckets for cell deposition on a 6-sq-cm area are described. Experimental results with different kinds of coating substances for glass slides as well as different isopyknic media are discussed, and data for differential cell counts are graphically demonstrated. Looking at the diagnostic accuracy and economic feasibility of this system, the authors realize that preparations have to be evaluated quantitatively and that constraints of sample size and processing time have to be taken into consideration for further developments.     

Perfect simulation from population genetic models with selection

We consider using the ancestral selection graph (ASG) to simulate samples from population genetic models with selection. Currently the use of the ASG to simulate samples is limited. This is because the computational requirement for simulating samples increases exponentially with the selection rate and also due to needing to simulate a sample of size one from the population at equilibrium. For the only case where the distribution of a sample of size one is known, that of parent-independent mutations, more efficient simulation algorithms exist. We will show that by applying the idea of coupling from the past to the ASG, samples can be simulated from a general K-allele model without knowledge of the distribution of a sample of size one. Furthermore, the computation involved in generating such samples appears to be less than that of simulating the ASG until its ultimate ancestor. In particular, in the case of genic selection with parent-independent mutations, the computational requirement increases only quadratically with the selection rate. The algorithm is demonstrated by simulating samples at a microsatellite locus.     

Quantum nature of photon signal emitted by Xanthoria parietina and its implications to biology

The properties of living systems are usually described in the semi-classical framework that makes phenomenological division of properties into four classes--matter, psyche, soft consciousness and hard consciousness. Quantum framework provides a scientific basis of this classification of properties. The scientific basis requires the existence of macroscopic quantum entity entangled with quantum photon field of a living system. Every living system emits a photon signal with features indicating its quantum nature. Quantum nature of the signal emitted by a sample of X. parietina is confirmed by analysing photo count distributions obtained in 20000 measurements of photon number in contiguous bins of sizes of 50, 100, 200, 300 and 500 ms. The measurements use a broadband detector sensitive in 300-800 nm range (Photo count distributions of background noise and observed signal are measured similarly. These measurements background noise corrected squeezed state parameters of the signal. The parameters are signal strength expressed in counts per bin, r = 0.06, theta = 2.76 and phi = 0.64. The parameters correctly reproduce photo count distribution of any bin size in 50 ms-6 s range. The reproduction of photo count distributions is a credible evidence of spontaneous emission of photon signal in a quantum squeezed state for macroscopic time by the sample. The evidence is extrapolated to other living systems emitting similar photon signals. It is suggested that every living system is associated with a photon field in squeezed state. The suggestion has far reaching implications to biology and provides two ways of observing and manipulating a living system--either through matter or field or a combination of the two. Some implications and possible scenarios are elaborated.     

种群微卫星DNA分析中样本量对各种遗传多样性度量指标的影响

我们以中国飞蝗种群的微卫星遗传分析数据为例 ,评估了取样对种群遗传多样性指标的影响 ,结果显示 :样本大小与所观测到的每位点等位基因数、平均等位基因数及基因丰富度指数均呈显著正相关 ,而与期望杂合度无显著相关 ;微卫星位点多态性的高低直接影响所观测到的种群基因丰富度及其检测所需的样本量 ;对大多数种群遗传和分子生态学研究而言 ,30 - 5 0个个体是微卫星DNA分析所需要的最小样本量。基因丰富度经过稀疏法或多次随机抽样法校正后 ,可适用于瓶颈效应等种群历史数量变动的检测。另外 ,在研究中 ,还应避免采集时间的不同及样本的性比构成所可能造成的对种群遗传结构的影响     

Measurement of marine picoplankton cell size by using a cooled, charge-coupled device camera with image-analyzed fluorescence microscopy.

Accurate measurement of the biomass and size distribution of picoplankton cells (0.2 to 2.0 microns) is paramount in characterizing their contribution to the oceanic food web and global biogeochemical cycling. Image-analyzed fluorescence microscopy, usually based on video camera technology, allows detailed measurements of individual cells to be taken. The application of an imaging system employing a cooled, slow-scan charge-coupled device (CCD) camera to automated counting and sizing of individual picoplankton cells from natural marine samples is described. A slow-scan CCD-based camera was compared to a video camera and was superior for detecting and sizing very small, dim particles such as fluorochrome-stained bacteria. Several edge detection methods for accurately measuring picoplankton cells were evaluated. Standard fluorescent microspheres and a Sargasso Sea surface water picoplankton population were used in the evaluation. Global thresholding was inappropriate for these samples. Methods used previously in image analysis of nanoplankton cells (2 to 20 microns) also did not work well with the smaller picoplankton cells. A method combining an edge detector and an adaptive edge strength operator worked best for rapidly generating accurate cell sizes. A complete sample analysis of more than 1,000 cells averages about 50 min and yields size, shape, and fluorescence data for each cell. With this system, the entire size range of picoplankton can be counted and measured.     

Comparative gas phase and pulsed liquid phase sequencing on a modified Applied Biosystems 477A sequencer

A simple and inexpensive modification of the Applied Biosystems 477A sequencer, to run in the pulsed liquid-phase and in the gas-phase mode of the Edman chemistry, is described. This modification is especially useful for sequencing samples on polyvinylidene difluoride (PVDF) membranes. Additional carriers are required if a sample on a PVDF membrane is sequenced with the pulsed liquid-phase degradation program of the 477A. In the gas-phase mode no such carriers are needed. This eliminates time-consuming preconditioning sequencer cycles and reduces the sequencer background. In addition, initial coupling yields in the gas-phase mode exceeded those in the pulsed liquid-phase mode, whereas the average repetitive yields were similar. Samples spotted onto glass fiber filters pretreated with polybrene and samples spotted or electroblotted onto PVDF membranes were examined. A number of advantages of the gas-phase mode are presented.     

Binomial sampling to estimate rust mite (Acari: Eriophyidae) densities on orange fruit

Binomial sampling based on the proportion of samples infested was investigated for estimating mean densities of citrus rust mite, Phyllocoptruta oleivora (Ashmead), and Aculops pelekassi (Keifer) (Acari: Eriophyidae), on oranges, Citrus sinensis (L.) Osbeck. Data for the investigation were obtained by counting the number of motile mites within 600 sample units (each unit a 1-cm2 surface area per fruit) across a 4-ha block of trees (32 blocks total): five areas per 4 ha, five trees per area, 12 fruit per tree, and two samples per fruit. A significant (r2 = 0.89), linear relationship was found between ln(-ln(1 -Po)) and ln(mean), where P0 is the proportion of samples with more than zero mites. The fitted binomial parameters adequately described a validation data set from a sampling plan consisting of 192 samples. Projections indicated the fitted parameters would apply to sampling plans with as few as 48 samples, but reducing sample size resulted in an increase of bootstrap estimates falling outside expected confidence limits. Although mite count data fit the binomial model, confidence limits for mean arithmetic predictions increased dramatically as proportion of samples infested increased. Binomial sampling using a tally threshold of 0 therefore has less value when proportions of samples infested are large. Increasing the tally threshold to two mites marginally improved estimates at larger densities. Overall, binomial sampling for a general estimate of mite densities seemed to be a viable alternative to absolute counts of mites per sample for a grower using a low management threshold such as two or three mites per sample.     

Measurement of marine picoplankton cell size by using a cooled, charge-coupled device camera with image-analyzed fluorescence microscopy.

Accurate measurement of the biomass and size distribution of picoplankton cells (0.2 to 2.0 microns) is paramount in characterizing their contribution to the oceanic food web and global biogeochemical cycling. Image-analyzed fluorescence microscopy, usually based on video camera technology, allows detailed measurements of individual cells to be taken. The application of an imaging system employing a cooled, slow-scan charge-coupled device (CCD) camera to automated counting and sizing of individual picoplankton cells from natural marine samples is described. A slow-scan CCD-based camera was compared to a video camera and was superior for detecting and sizing very small, dim particles such as fluorochrome-stained bacteria. Several edge detection methods for accurately measuring picoplankton cells were evaluated. Standard fluorescent microspheres and a Sargasso Sea surface water picoplankton population were used in the evaluation. Global thresholding was inappropriate for these samples. Methods used previously in image analysis of nanoplankton cells (2 to 20 microns) also did not work well with the smaller picoplankton cells. A method combining an edge detector and an adaptive edge strength operator worked best for rapidly generating accurate cell sizes. A complete sample analysis of more than 1,000 cells averages about 50 min and yields size, shape, and fluorescence data for each cell. With this system, the entire size range of picoplankton can be counted and measured.     

Detection of mutations in PCR products from clinical samples by surface plasmon resonance

Two different strategies for scanning and screening of mutations in polymerase chain reaction (PCR) products by hybridization analysis are described, employing real-time biospecific interaction analysis (BIA) for detection. Real-time BIA was used to detect differences in hybridization responses between PCR products and different 17-mer oligonucleotide probes. For the analysis using a biosensor instrument, two different experimental formats were investigated based on immobilization of either biotinylated PCR products or oligonucleotide probes onto a sensor chip. Applied on the human tumour suppressor p53 gene, differences in hybridization levels for full-match and mismatch situations employing both formats allowed the detection of point mutations in exon 6 PCR products, derived from a breast tumour biopsy sample. In addition, a mutant sample sequence could be detected in a 50/50 background of wild type exon 6 sequence. The suitability of the different formats for obtaining a regenerable system and a high throughput of samples is discussed. © 1997 John Wiley & Sons, Ltd.     

Determination of size and charge distributions by combination of quasi-elastic light scattering and band transport

A convenient method for the determination of size and charge distributions of polydisperse samples is described. It includes three steps: (a) band transport to disperse the components of the sample in a scattering cell; (b) total intensity scattering to determine the concentration and mobility at each location; and (c) quasi-elastic light scattering (QLS) to characterize the hydrodynamic sizes. Combining the results of (b) and (c) yields size and charge distributions. Since complete resolution of the various components of the sample cannot occur in step (a), a method is described to estimate the average form factor, from the local mean size and the local degree of polydispersity obtained by QLS. The potential of this method is demonstrated by the results of its application to two radiocolloids which have broad size distribution (50- to 600-nm diameters).     

Automated in-solution protein digestion using a commonly available high-performance liquid chromatography autosampler

A completely automated peptide mapping liquid chromatography/mass spectrometry (LC/MS) system for characterization of therapeutic proteins in which a common high-performance liquid chromatography (HPLC) autosampler is used for automated sample preparation, including protein denaturation, reduction, alkylation, and enzymatic digestion, is described. The digested protein samples are then automatically subjected to LC/MS analysis using the same HPLC system. The system was used for peptide mapping of monoclonal antibodies (mAbs), known as a challenging group of therapeutic proteins for achieving complete coverage and quantitative representation of all peptides. Detailed sample preparation protocols, using an Agilent HPLC system, are described for Lys-C digestion of mAbs with intact disulfide bonds and tryptic digestion of mAbs after reduction and alkylation. The automated procedure of Lys-C digestion of nonreduced antibody, followed by postdigestion disulfide reduction, produces both the nonreduced and reduced digests that facilitate disulfide linkage analysis. The automated peptide mapping LC/MS system has great utility in preparing and analyzing multiple samples for protein characterization, identification, and quantification of posttranslational modifications during process and formulation development as well as for protein identity and quality control.     

Sample size and sampling error in geometric morphometric studies of size and shape

Geometric morphometric studies are increasingly becoming common in systematics and palaeontology. The samples in such studies are often small, due to the paucity of material available for analysis. However, very few studies have tried to assess the impact of sampling error on analytical results. Here, this issue is addressed empirically using repeated randomized selection experiments to build progressively smaller samples from an original dataset of ∼400 vervet monkey (Cercopithecus aethiops) skulls. Size and shape parameters (including mean size and shape, size and shape variances, angles of allometric trajectories) that are commonly used in geometric morphometric studies, are estimated first in the original sample and then in the random subsamples. Estimates are then compared to give an indication of what is the minimum desirable sample size for each parameter. Mean size, standard deviation of size and variance of shape are found to be fairly accurate even in relatively small samples. In contrast, mean shapes and angles between static allometric trajectories are strongly affected by sampling error. If confirmed in other groups, our findings may have substantial implications for studies of morphological variation in present and fossil species. By performing rarefaction analyses like those presented in our study, morphometricians can be easily provided with important clues on how a simple but crucial factor like sample size can alter results of their studies.