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1.
The performance of sera pre-treatments for biomarker discovery has been recently assessed as very poor not only for immuno-subtraction, in turn evaluated as a tool unable to look deep into the low-abundance proteome (LAP) and thus incapable to lead to any novel biomarker discovery (J Proteome Res 2010;9:4982-4991), but also for combinatorial peptide ligand libraries (CPLL) (Proteomics 2010;10:1416-1425). The performance of both tools has been given as enabling to barely detect a meagre 25% more as compared to control, untreated sera. Meanwhile, other studies indicated the extreme effectiveness of peptide libraries to enlarge the knowledge of proteome compositions. In this contradictory situation we are here re-evaluating some protocol aspects and report that indeed CPLL is an excellent tool, able to dig really deep into the low-abundance proteome. The problem is that in those reports under-optimized capture and elution protocols had been adopted. With the protocols here reported, namely (a) abandoning the step of adding 150mM salt to the sample; (b) capture at three different pH values (pH 4.0, 7.0 and 9.3) and (c), most importantly, eluting from CPLL beads in 4% boiling SDS in the presence of 25mM DTT, we can largely expand the windows of visibility. In particular, it is here shown that a common elution protocol adopted in several reports, in 4M urea and 1% CHAP, barely elutes about 15% of the captured species. Nevertheless if the CPLL beads thus treated are further eluted with boiling SDS-DTT, an additional 80% is recovered. 相似文献
2.
Elisa Fasoli Alessia Farinazzo Cheng Jun Sun Alexander V. Kravchuk Luc Guerrier Frederic Fortis Egisto Boschetti Pier Giorgio Righetti 《Journal of Proteomics》2010,73(4):733-742
When capturing proteins via combinatorial peptide ligand libraries, a method well known for drastically reducing the concentration of high-abundance proteins and substantially magnifying the signal of low-abundance species, thus leading to the discovery of a large number of proteins previously undetected in proteomes, we had constantly noticed that there would be a loss of species initially present in the untreated sample, to the tune of 5%, up to 15% in some cases. Such losses are a nuisance and hamper to some extent the unique performance of the method. In order to verify if such losses could be reduced and also to understand some mechanisms of the capture process, we introduce here an important variant to the capture operation, up to the present carried out in physiological saline at pH 7.2. In this novel protocol, the binding step is conducted at three different pH values, namely the standard one at pH 7.2, plus two additional processes, at acidic (pH 4.0) and alkaline (pH 9.3) pH values. Indeed the capture process is more extensive, with a number of additional species captured at the two pH extremes in sera and other proteomes. Interestingly, at pH 4.0 newly detected proteins were mostly acidic, while at the alkaline pH additional protein species were more evenly distributed throughout the pI range towards the alkaline area. The role of pH in the complex mechanism of binding among the hexapeptide library and the various proteomes being analyzed is discussed and evaluated. Due to significant changes in protein patterns with pH, recommendations are thus made to increase the possibility to find additional gene products illustrated by two examples (snake venom and leaf protein extract). Keeping under control the environmental pH when facing reproducibility studies or for comparative proteomics profiling is also a general rule suggested by this study. 相似文献
3.
A critical step in protein biomarker discovery is the ability to contrast proteomes, a process referred generally as quantitative proteomics. While stable-isotope labeling (e.g., ICAT, 18O- or 15N-labeling, or AQUA) remains the core technology used in mass spectrometry-based proteomic quantification, increasing efforts have been directed to the label-free approach that relies on direct comparison of peptide peak areas between LC-MS runs. This latter approach is attractive to investigators for its simplicity as well as cost effectiveness. In the present study, the reproducibility and linearity of using a label-free approach to highly complex proteomes were evaluated. Various amounts of proteins from different proteomes were subjected to repeated LC-MS analyses using an ion trap or Fourier transform mass spectrometer. Highly reproducible data were obtained between replicated runs, as evidenced by nearly ideal Pearson's correlation coefficients (for ion's peak areas or retention time) and average peak area ratios. In general, more than 50% and nearly 90% of the peptide ion ratios deviated less than 10% and 20%, respectively, from the average in duplicate runs. In addition, the multiplicity ratios of the amounts of proteins used correlated nicely with the observed averaged ratios of peak areas calculated from detected peptides. Furthermore, the removal of abundant proteins from the samples led to an improvement in reproducibility and linearity. A computer program has been written to automate the processing of data sets from experiments with groups of multiple samples for statistical analysis. Algorithms for outlier-resistant mean estimation and for adjusting statistical significance threshold in multiplicity of testing were incorporated to minimize the rate of false positives. The program was applied to quantify changes in proteomes of parental and p53-deficient HCT-116 human cells and found to yield reproducible results. Overall, this study demonstrates an alternative approach that allows global quantification of differentially expressed proteins in complex proteomes. The utility of this method to biomarker discovery is likely to synergize with future improvements in the detecting sensitivity of mass spectrometers. 相似文献
4.
The present review highlights recent progresses in the technique of combinatorial peptide ligand libraries (CPPL), a methodology that has much to offer for the detection of low- to very-low abundance proteins (nanograms/mL scale and below) in any proteome. In particular, advances in exploration of the urinary, plasma and tissue proteomes are discussed and evaluated. It is shown that when treating biological fluids, such as plasma, with CPLLs, the detection sensitivity, which in the control only reaches 10 ng/mL, can be enhanced to as high as 10 pg/mL, with an increment of sensitivity of three orders of magnitude. The possibility of using CPLLs as a two-dimensional pre-fractionation of any proteome is also evaluated: on the charge axis, CPLL capture can be implemented at no less than three different pH values (4.0, 7.2 and 9.3), thus permitting a capture of proteinaceous analytes bearing a net positive or net negative charge, respectively. When capture is performed in the absence of salts or at high levels of salts (of the Hofmeister series), one can favor the capture of hydrophilic vs. hydrophobic proteins, respectively. This would thus be a genuine 2D protocol, working on orthogonal separation principles (charge vs. hydrophobicity). As the horizon of CPLLs is expanding and its use is exponentially growing, we expect major breakthroughs in, e.g., biomarker discovery, a field that has suffered a decade of failures. 相似文献
5.
《Expert review of proteomics》2013,10(1):51-65
Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, or surface-enhanced laser desorption/ionization ProteinChip® technology, has been widely used in obtaining the quantitative profiles of tissue proteomes, particularly plasma proteomes. Its high-throughput nature and simplicity in its experimental procedures have allowed this technology to become a popular research tool for biomarker discovery in the past 5 years. After accumulating more research experiences, researchers now have a better understanding of the characteristics and limitations of this technology, as well as the pitfalls in biomarker research, by undertaking a comparative proteomic approach. This review provides an overview of the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, discusses its limitations and provides some possible solutions to help apply this technology to biomarker research. 相似文献
6.
Poon TC 《Expert review of proteomics》2007,4(1):51-65
Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, or surface-enhanced laser desorption/ionization ProteinChip technology, has been widely used in obtaining the quantitative profiles of tissue proteomes, particularly plasma proteomes. Its high-throughput nature and simplicity in its experimental procedures have allowed this technology to become a popular research tool for biomarker discovery in the past 5 years. After accumulating more research experiences, researchers now have a better understanding of the characteristics and limitations of this technology, as well as the pitfalls in biomarker research, by undertaking a comparative proteomic approach. This review provides an overview of the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, discusses its limitations and provides some possible solutions to help apply this technology to biomarker research. 相似文献
7.
Maria Pernemalm Luigi De Petris Hanna Eriksson Eva Brandén Hirsh Koyi Lena Kanter Rolf Lewensohn Janne Lehtiö 《Proteomics》2009,9(13):3414-3424
In this study we applied narrow‐range peptide IEF to plasma or pleural effusion prior to LC/MS/MS. Two methods for narrow‐range IEF were run; IPG strips and free‐flow electrophoresis. Data from this study was compared with cell line data to evaluate the method performance in body fluids. To test the methods potential in quantitative biomarker discovery studies, plasma and pleural effusion from patients with lung adenocarcinoma (n=3) were compared with inflammatory pleuritis (n=3) using iTRAQ quantification. Using narrow‐range IEF on the peptide level we were able to identify and quantify 282 proteins in plasma and 300 proteins in pleural effusion. These body fluid proteomes demonstrated high degree of overlap; however, more proteins significantly differently altered levels related to adenenocarcinoma were found in pleural effusion compared with plasma, suggesting enrichment of lung tissue‐related proteins in pleural effusion. Nine proteins were chosen for initial validation with Western blot, and one protein (NPC2) was chosen for further validation using imunohistochemistry. Overall, the quantitative results from IEF/LC/MS/MS showed good correlation with the results from Western blot and imunohistochemistry, showing the potential of this methodology in quantitative biomarker discovery studies. 相似文献
8.
Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers 总被引:4,自引:0,他引:4
Whiteaker JR Zhao L Zhang HY Feng LC Piening BD Anderson L Paulovich AG 《Analytical biochemistry》2007,362(1):44-54
A major bottleneck for validation of new clinical diagnostics is the development of highly sensitive and specific assays for quantifying proteins. We previously described a method, stable isotope standards with capture by antipeptide antibodies, wherein a specific tryptic peptide is selected as a stoichiometric representative of the protein from which it is cleaved, is enriched from biological samples using immobilized antibodies, and is quantitated using mass spectrometry against a spiked internal standard to yield a measure of protein concentration. In this study, we optimized a magnetic-bead-based platform amenable to high-throughput peptide capture and demonstrated that antibody capture followed by mass spectrometry can achieve ion signal enhancements on the order of 10(3), with precision (CVs <10%) and accuracy (relative error approximately 20%) sufficient for quantifying biomarkers in the physiologically relevant ng/mL range. These methods are generally applicable to any protein or biological fluid of interest and hold great potential for providing a desperately needed bridging technology between biomarker discovery and clinical application. 相似文献
9.
Candiano G Santucci L Bruschi M Petretto A D'Ambrosio C Scaloni A Righetti PG Ghiggeri GM 《Journal of Proteomics》2012,75(3):796-805
A new method is here reported for facile elution of the human urinary proteome after being captured with combinatorial peptide ligand libraries (CPLL, ProteoMiner). It consists in challenging the beads with 100 mM Tris, pH 7.4, or with 100 mM Lys, pH 7.4 or even better with a mixture of Lys, Arg, Asp and Glu (150 mM final concentration). These elutions permit recovery of species in a native form, for monitoring any biological activity of the eluted species, while avoiding the noxious presence of sodium dodecyl sulphate (SDS), reported as the best eluant so far from CPLL beads. SDS, albeit permitting quantitative recovery from the beads, has to be removed from the sample prior to mass spectrometry analysis. This unorthodox elution, which most likely will work only for urine samples, seems to be due to the fact that bile salts and urinary pigments are massively adsorbed by the beads, thus masking the hydrophobic binding sites of aromatic and non-aromatic amino acids. The binding thus occurs mostly via ionic and hydrogen bond interactions via the “Grand Catchers” Arg, Lys, His, which can then be easily challenged by positively charged species, such a Tris, free Lys and free Arg in the eluant as well as by negatively charged compounds, such as Glu and Asp. When eluting with the four-amino acid mix, at least 3300 spots can be visualized in a 2D map. 相似文献
10.
Serum/Plasma depletion with chicken immunoglobulin Y antibodies for proteomic analysis from multiple Mammalian species. 总被引:3,自引:0,他引:3
Douglas Hinerfeld David Innamorati John Pirro Sun W Tam 《Journal of biomolecular techniques》2004,15(3):184-190
Plasma from different species is the most accessible and valuable source for biomarker discovery in clinical and animal samples. However, due to the high abundance of some proteins such as albumin and immunoglobulins, low-abundant proteins are often undetectable in proteomic analysis of plasma. We have established a plasma depletion scheme using chicken antibodies against various abundant proteins. This immunoaffinity purification procedure is able to deplete albumin across multiple species. The high binding capacity and specificity of the chicken antibody enables the efficient capture of its ligand from microliter volumes of plasma sample. The resulting two-dimensional gel analyses of the depleted and captured samples show significant enhancement of the low-abundant proteins and specific capture of the abundant ligand. By utilizing this sample preparation scheme, it is now possible to analyze the plasma proteome from multiple species in a potentially rapid and large-scale capacity for biomarker discovery, drug target discovery, and toxicology studies. 相似文献
11.
Song X Bandow J Sherman J Baker JD Brown PW McDowell MT Molloy MP 《Journal of proteome research》2008,7(7):2952-2958
There is considerable interest in using mass spectrometry for biomarker discovery in human blood plasma. We investigated aspects of experimental design for large studies that require analysis of multiple sample sets using iTRAQ reagents for sample multiplexing and quantitation. Immunodepleted plasma samples from healthy volunteers were compared to immunodepleted plasma from patients with osteoarthritis in eight separate iTRAQ experiments. Our analyses utilizing ProteinPilot software for peptide identification and quantitation showed that the methodology afforded excellent reproducibility from run to run for determining protein level ratios (coefficient of variation 11.7%), in spite of considerable quantitative variances observed between different peptides for a given protein. Peptides with high variances were associated with lower intensity iTRAQ reporter ions, while immunodepletion prior to sample analysis had a negligible affect on quantitative variance. We examined the influence of different reference samples, such as pooled samples or individual samples on calculating quantitative ratios. Our findings are discussed in the context of optimizing iTRAQ experimental design for robust plasma-based biomarker discovery. 相似文献
12.
Ronald J. Holewinski Zhicheng Jin Matthew J. Powell Matthew D. Maust Jennifer E. Van Eyk 《Proteomics》2013,13(5):743-750
Analysis of serum and plasma proteomes is a common approach for biomarker discovery, and the removal of high‐abundant proteins, such as albumin and immunoglobins, is usually the first step in the analysis. However, albumin binds peptides and proteins, which raises concerns as to how the removal of albumin could impact the outcome of the biomarker study while ignoring the possibility that this could be a biomarker subproteome itself. The first goal of this study was to test a new commercially available affinity capture reagent from Protea Biosciences and to compare the efficiency and reproducibility to four other commercially available albumin depletion methods. The second goal of this study was to determine if there is a highly efficient albumin depletion/isolation system that minimizes sample handling and would be suitable for large numbers of samples. Two of the methods tested (Sigma and ProteaPrep) showed an albumin depletion efficiency of 97% or greater for both serum and cerebrospinal fluid (CSF). Isolated serum and CSF albuminomes from ProteaPrep spin columns were analyzed directly by LC‐MS/MS, identifying 128 serum (45 not previously reported) and 94 CSF albuminome proteins (17 unique to the CSF albuminome). Serum albuminome was also isolated using Vivapure anti‐HSA columns for comparison, identifying 105 proteins, 81 of which overlapped with the ProteaPrep method. 相似文献
13.
For a long time, targeted and discovery proteomics covered different corners of the detection spectrum, with targeted proteomics focused on small target sets. This changed with the recent advances in highly multiplexed analysis. While discovery proteomics still pushes higher numbers of identified and quantified proteins, the advances in targeted proteomics rose to cover large parts of less complex proteomes or proteomes with low protein detection counts due to dynamic range restrictions, like the blood proteome. These new developments will impact, especially on the field of biomarker discovery and the possibility of using targeted proteomics for diagnostic purposes. 相似文献
14.
Slebos RJ Brock JW Winters NF Stuart SR Martinez MA Li M Chambers MC Zimmerman LJ Ham AJ Tabb DL Liebler DC 《Journal of proteome research》2008,7(12):5286-5294
Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (< 100 microg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 microg) and small (10 microg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 microg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 microg) samples. In nine replicate analyses of tryptic peptides from 50 microg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens. 相似文献
15.
Today biomarker discovery is one of the most active aspects of proteomic investigations. However, the wide dynamic range of plasma proteins makes the analysis very challenging because high abundance proteins tend to mask those of lower abundance. Using a large bead-based library of combinatorial peptide ligands (Equalizer beads or ProteoMiner), the dynamic range of the protein concentration is compressed, the high abundance proteins present in the sample are reduced and the low abundance proteins are enriched, while retaining representatives of all proteins within the sample. In the present study, the combination of beads with surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and two-dimensional differential gel electrophoresis (2-D DIGE) technology were evaluated considering efficiency, reproducibility, sensitivity, and compatibility. The bead technology is easily compatible with both SELDI-TOF-MS and 2-D DIGE and the samples can be analyzed directly without any processing of the sample. The use of the beads prior SELDI-TOF-MS and 2-D DIGE enabled detection of many new protein spots/peaks and increased resolution and improved intensity of low abundance proteins in a reproducible fashion compared with the depletion technique. Several proteins have been identified by the combination of beads, 2-D DIGE and MS for example different kinds of complement factors and cytoskeletal proteins. Our data suggest that integration of the bead technology with our current proteomic technologies will enhance the possibility to deliver new peptide/protein biomarker candidates in our projects. 相似文献
16.
Jagannath Swaminathan Alexander A. Boulgakov Edward M. Marcotte 《PLoS computational biology》2015,11(2)
The proteomes of cells, tissues, and organisms reflect active cellular processes and change continuously in response to intracellular and extracellular cues. Deep, quantitative profiling of the proteome, especially if combined with mRNA and metabolite measurements, should provide an unprecedented view of cell state, better revealing functions and interactions of cell components. Molecular diagnostics and biomarker discovery should benefit particularly from the accurate quantification of proteomes, since complex diseases like cancer change protein abundances and modifications. Currently, shotgun mass spectrometry is the primary technology for high-throughput protein identification and quantification; while powerful, it lacks high sensitivity and coverage. We draw parallels with next-generation DNA sequencing and propose a strategy, termed fluorosequencing, for sequencing peptides in a complex protein sample at the level of single molecules. In the proposed approach, millions of individual fluorescently labeled peptides are visualized in parallel, monitoring changing patterns of fluorescence intensity as N-terminal amino acids are sequentially removed, and using the resulting fluorescence signatures (fluorosequences) to uniquely identify individual peptides. We introduce a theoretical foundation for fluorosequencing and, by using Monte Carlo computer simulations, we explore its feasibility, anticipate the most likely experimental errors, quantify their potential impact, and discuss the broad potential utility offered by a high-throughput peptide sequencing technology. 相似文献
17.
Kulasingam V Smith CR Batruch I Buckler A Jeffery DA Diamandis EP 《Journal of proteome research》2008,7(2):640-647
While numerous strategies exist for biomarker discovery, the bottleneck to product development and routine use at the clinic is in the verification phase of candidate biomarkers. The aim of this study was to establish a robust and high-throughput product ion monitoring (PIM) assay that is potentially capable of rapidly verifying candidates from discovery phase experiments. Using prostate-specific antigen (PSA), a model biomarker, and a routinely used mass spectrometer for discovery platforms, an ion trap (LTQ, Thermo), the utility of this instrument to perform PIM was explored. The proteotypic doubly charged intact peptide LSEPAELTDAVK ( m/ z 637) fragmenting to m/ z 943 (PAELTDAVK) was monitored. A limit of detection of 10 attomoles with a coefficient of variation (CV) of <20% was obtained for a purified recombinant PSA digest. Immunoextraction of endogenous PSA from serum using a monoclonal antibody on a 96-well microtiter plate, followed by PIM on the LTQ, enabled quantification of PSA down to less than 1 ng/mL with a limit of detection of 0.1 ng/mL and CVs < 20%. Mascot searching and ion ratio confirmation further supported the conclusion that the quantified moiety in serum was the PSA peptide. We conclude that this methodology could be adapted quickly and easily to other candidates, thus providing a much needed technology to bridge the gap between discovery and validation platforms. 相似文献
18.
《Expert review of proteomics》2013,10(4):337-354
Despite intense global efforts, no new clinical and/or viable biomarkers have been established to overcome the limitation of the prostate specific antigen in the early diagnosis and prognosis of prostate cancer (PCa). The current proteomic approaches to PCa biomarker discovery, each have distinct advantages and disadvantages, yet when combined hold real promise in the coming years. One key approach to this effort is the development of non-targeted, depletion-free and quantitative liquid chromatography–ultra high resolution tandem mass spectrometry (LC–MS) pipelines for the systems-wide interrogation of the diverse proteomes encompassed in whole tissue and blood serum or plasma. Derived quantitative proteomes can be decoded for their biomedical relevance with advanced bioinformatics and bibliographic mining to yield promising ‘molecular portraits’ that can gauge prostatic disease at the serological level. Their functional annotation, although potentially useful, is beyond our current level of biological understanding and should not be requisite for their effective use in the clinical monitoring of prostatic disease. 相似文献
19.
Beer LA Tang HY Sriswasdi S Barnhart KT Speicher DW 《Journal of proteome research》2011,10(3):1126-1138
Ectopic pregnancy (EP) and normal intrauterine pregnancy (IUP) serum proteomes were quantitatively compared to systematically identify candidate biomarkers. A 3-D biomarker discovery strategy consisting of abundant protein immunodepletion, SDS gels, LC-MS/MS, and label-free quantitation of MS signal intensities identified 70 candidate biomarkers with differences between groups greater than 2.5-fold. Further statistical analyses of peptide quantities were used to select the most promising 12 biomarkers for further study, which included known EP biomarkers, novel EP biomarkers (ADAM12 and ISM2), and five specific isoforms of the pregnancy specific beta-1-glycoprotein family. Technical replicates showed good reproducibility and protein intensities from the label-free discovery analysis compared favorably with reported abundance levels of several known reference serum proteins over at least 3 orders of magnitude. Similarly, relative abundances of candidate biomarkers from the label-free discovery analysis were consistent with relative abundances from pilot validation assays performed for five of the 12 most promising biomarkers using label-free multiple reaction monitoring of both the patient serum pools used for discovery and the individual samples that constituted these pools. These results demonstrate robust, reproducible, in-depth 3-D serum proteome discovery, and subsequent pilot-scale validation studies can be achieved readily using label-free quantitation strategies. 相似文献
20.
Shotgun proteomic methods involving iTRAQ (isobaric tags for relative and absolute quantitation) peptide labeling facilitate quantitative analyses of proteomes and searches for useful biomarkers. However, the plasma proteome''s complexity and the highly dynamic plasma protein concentration range limit the ability of conventional approaches to analyze and identify a large number of proteins, including useful biomarkers. The goal of this paper is to elucidate the best approach for plasma sample pretreatment for MS- and iTRAQ-based analyses. Here, we systematically compared four approaches, which include centrifugal ultrafiltration, SCX chromatography with fractionation, affinity depletion, and plasma without fractionation, to reduce plasma sample complexity. We generated an optimized protocol for quantitative protein analysis using iTRAQ reagents and an UltrafleXtreme (Bruker Daltonics) MALDI TOF/TOF mass spectrometer. Moreover, we used a simple, rapid, efficient, but inexpensive sample pretreatment technique that generated an optimal opportunity for biomarker discovery. We discuss the results from the four sample pretreatment approaches and conclude that SCX chromatography without affinity depletion is the best plasma sample preparation pretreatment method for proteome analysis. Using this technique, we identified 1,780 unique proteins, including 1,427 that were quantified by iTRAQ with high reproducibility and accuracy. 相似文献