Robust MS quantification method for phospho-peptides using 18O/16O labeling

Background  

Quantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using ¹⁸O/¹⁶O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-β treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable.     

Development of a novel method for quantification of autophagic protein degradation by AHA labeling

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.     

Development of a novel method for quantification of autophagic protein degradation by AHA labeling

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.     

In-gel stable isotope labeling for relative quantification using mass spectrometry

Although differences in protein staining intensity can often be visualized by difference gel electrophoresis, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. We present a protocol for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using in-gel stable isotope labeling. In this protocol protein extracts from any source treated under two experimental conditions are resolved in two separate lanes by gel electrophoresis. Parallel gel regions of interest are reacted separately with either light or heavy isotope-labeled reagents, and the gel slices are then combined and digested with proteases. The resulting peptides are then analyzed by liquid chromatography/mass spectrometry (LC/MS) to determine relative abundance of light- and heavy-isotope lysine-containing peptide pairs and analyzed by LC/MS/MS for identification of sequence and modifications. This protocol should take approximately 24-26 h to complete, including the incubation time for proteolytic digestion. Additional time will be needed for data analysis and interpretation.     

New selective method for quantification of organosilanol groups in silicone pre-elastomers

The silicone elastomers used for drug delivery are normally reinforced by fumed silica, which contains a high density of silanol groups. These inorganic silanol groups have to be deactivated in order to avoid stiffening the uncured pre-elastomer, also called creep hardening. One commonly used way of achieving this deactivation is to mix the material with low molecular mass organosilanols at an elevated temperature. It is important to be able to quantify the nonbonded organosilanols remaining in the material after manufacture. Traditional testing does not distinguish between inorganic silanols and organosilanols. A new selective method for the quantification of organosilanol groups in silicone pre-elastomers has therefore been developed. This method is based on derivatization of the silanol groups with a mixture of dimethylphenylchlorosilane and tetramethyldiphenylsilazane, so that the silanol groups are replaced with a dimethylphenyl group. The derivatized organosilanols are then determined by liquid chromatography using a size exclusion column and a UV detector. No interference was found from other groups normally present in medical grade pre-elastomers, such as vinyls, hydrides, and inorganic bonded silanol on silica or water. The results agreed well with the nonselective Karl Fischer titration for some short chain silanols.     

A method for bacteriocin quantification

Different aspects of the most commonly used assay methods in the study of bacteriocins were examined. The conditions under which extraction and incubation (including exposure time) take place were analysed, and several different formal models that are usually employed to calculate ID50 were compared. As an alternative designed to overcome the problems which characterize the response of micro-organisms that are sensitive to bacteriocins, an operative procedure in a liquid medium and a modified re-parameterized logistic equation is proposed. When applied to the inhibition of Leuconostoc mesenteroides by nisin, the model allows an optimal experimental procedure to be defined.     

An accurate proteomic quantification method: Fluorescence labeling absolute quantification (FLAQ) using multidimensional liquid chromatography and tandem mass spectrometry

A facile proteomic quantification method, fluorescent labeling absolute quantification (FLAQ), was developed. Instead of using MS for quantification, the FLAQ method is a chromatography-based quantification in combination with MS for identification. Multidimensional liquid chromatography (MDLC) with laser-induced fluorescence (LIF) detection with high accuracy and tandem MS system were employed for FLAQ. Several requirements should be met for fluorescent labeling in MS identification: Labeling completeness, minimum side-reactions, simple MS spectra, and no extra tandem MS fragmentations for structure elucidations. A fluorescence dye, 5-iodoacetamidofluorescein, was finally chosen to label proteins on all cysteine residues. The fluorescent dye was compatible with the process of the trypsin digestion and MALDI MS identification. Quantitative labeling was achieved with optimization of reacting conditions. A synthesized peptide and model proteins, BSA (35 cysteines), OVA (five cysteines), were used for verifying the completeness of labeling. Proteins were separated through MDLC and quantified based on fluorescent intensities, followed by MS identification. High accuracy (RSD% < 1.58) and wide linearity of quantification (1-10(5) ) were achieved by LIF detection. The limit of quantitation for the model protein was as low as 0.34 amol. Parts of proteins in human liver proteome were quantified and demonstrated using FLAQ.     

Development of a bead-based multiplex genotyping method for diagnostic characterization of HPV infection

The accurate genotyping of human papillomavirus (HPV) is clinically important because the oncogenic potential of HPV is dependent on specific genotypes. Here, we described the development of a bead-based multiplex HPV genotyping (MPG) method which is able to detect 20 types of HPV (15 high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 5 low-risk HPV types 6, 11, 40, 55, 70) and evaluated its accuracy with sequencing. A total of 890 clinical samples were studied. Among these samples, 484 were HPV positive and 406 were HPV negative by consensus primer (PGMY09/11) directed PCR. The genotyping of 484 HPV positive samples was carried out by the bead-based MPG method. The accuracy was 93.5% (95% CI, 91.0-96.0), 80.1% (95% CI, 72.3-87.9) for single and multiple infections, respectively, while a complete type mismatch was observed only in one sample. The MPG method indiscriminately detected dysplasia of several cytological grades including 71.8% (95% CI, 61.5-82.3) of ASCUS (atypical squamous cells of undetermined significance) and more specific for high grade lesions. For women with HSIL (high grade squamous intraepithelial lesion) and SCC diagnosis, 32 women showed a PPV (positive predictive value) of 77.3% (95% CI, 64.8-89.8). Among women >40 years of age, 22 women with histological cervical cancer lesions showed a PPV of 88% (95% CI, 75.3-100). Of the highest risk HPV types including HPV-16, 18 and 31 positive women of the same age groups, 34 women with histological cervical cancer lesions showed a PPV of 77.3% (95% CI, 65.0-89.6). Taken together, the bead-based MPG method could successfully detect high-grade lesions and high-risk HPV types with a high degree of accuracy in clinical samples.     

TRICORE, a novel bead-based specimen concentration method for the culturing of Mycobacterium tuberculosis

Centrifugation is a necessary concentrating step for the detection of Mycobacterium tuberculosis in a liquid culture. However, centrifugation is biologically hazardous and presents an obstacle in the development of an automated culture system. A bead-based bacterial concentration method, TRICORE, was recently developed by Genetein Co., Ltd. We compared the efficacy of TRICORE and conventional centrifugation for concentrating M. tuberculosis in clinical sputum specimens by using liquid and solid culture systems. Among 90 pretreated clinical sputum specimens, 51 (57.3%) and 55 (61.8%) M. tuberculosis isolates were recovered by the MGIT culture system by using the centrifugation and TRICORE methods, respectively (chi-square test, p=0.5413). The detection time for the centrifugation method was 359.3±117.0 h, while that for the bead-based concentration method was 377.6±162.3 h (p=0.5637). However, the number of colonies recovered on solid media were significantly higher with the TRICORE method (p=0.003). In particular, among the smear-negative specimens, culture positivity of the TRICORE method was 39.6%, while that of the centrifugation method was 15.1%. The TRICORE bead-based concentration method was considered equivalent to centrifugation and enabled efficient collection of paucibacillary specimens in solution. Thus, the new noncentrifugation concentration method could yield more positive culture results.     

Magnetic bead-based DNA detection with multi-layers quantum dots labeling for rapid detection of Escherichia coli O157:H7

This work demonstrated the feasibility of detecting 250zM Escherichia coli O157:H7 eaeA target DNA by using a magnetic bead-based DNA detection assay with designed labeling strategy within 40-60min. The magnetic beads were used as the solid support for the binding probe and isolated the target DNA from the sample. The detection signals could be amplified from the multi-layers biotin-streptavidin conjugated quantum dots based on binding with specific designed biotinlyted linker. This assay method would provide a simple, rapid, and ultra-sensitive detection method for DNA or other biomolecular analysis.     

A bead-based method for multiplexed identification and quantitation of DNA sequences using flow cytometry

A new multiplexed, bead-based method which utilizes nucleic acid hybridizations on the surface of microscopic polystyrene spheres to identify specific sequences in heterogeneous mixtures of DNA sequences is described. The method consists of three elements: beads (5.6-microm diameter) with oligomer capture probes attached to the surface, three fluorophores for multiplexed detection, and flow cytometry instrumentation. Two fluorophores are impregnated within each bead in varying amounts to create different bead types, each associated with a unique probe. The third fluorophore is a reporter. Following capture of fluorescent cDNA sequences from environmental samples, the beads are analyzed by flow cytometric techniques which yield a signal intensity for each capture probe proportional to the amount of target sequences in the analyte. In this study, a direct hybrid capture assay was developed and evaluated with regard to sequence discrimination and quantitation of abundances. The target sequences (628 to 728 bp in length) were obtained from the 16S/23S intergenic spacer region of microorganisms collected from polluted groundwater at the nuclear waste site in Hanford, Wash. A fluorescence standard consisting of beads with a known number of fluorescent DNA molecules on the surface was developed, and the resolution, sensitivity, and lower detection limit for measuring abundances were determined. The results were compared with those of a DNA microarray using the same sequences. The bead method exhibited far superior sequence discrimination and possesses features which facilitate accurate quantitation.     

High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing

Small RNA cloning and sequencing is uniquely positioned as a genome-wide approach to quantify miRNAs with single-nucleotide resolution. However, significant biases introduced by RNA ligation in current protocols lead to inaccurate miRNA quantification by 1000-fold. Here we report an RNA cloning method that achieves over 95% efficiency for both 5′ and 3′ ligations. It achieves accurate quantification of synthetic miRNAs with less than two-fold deviation from the anticipated value and over a dynamic range of four orders of magnitude. Taken together, this high-efficiency RNA cloning method permits accurate genome-wide miRNA profiling from total RNAs.     

Sortagging: a versatile method for protein labeling

Genetically encoded reporter constructs that yield fluorescently labeled fusion proteins are a powerful tool for observing cell biological phenomena, but they have limitations. Sortagging (sortase-mediated transpeptidation) is a versatile chemoenzymatic system for site-specific labeling of proteins with small (<2 kDa) probes. Sortagging combines the precision of a genetically encoded tag with the specificity of an enzymatic reaction and the ease and chemical versatility of peptide synthesis. Here we apply this technique to proteins in vitro and on the surface of living cells.     

A fast ab-initio method for predicting miRNA precursors in genomes

miRNAs are small non coding RNA structures which play important roles in biological processes. Finding miRNA precursors in genomes is therefore an important task, where computational methods are required. The goal of these methods is to select potential pre-miRNAs which could be validated by experimental methods. With the new generation of sequencing techniques, it is important to have fast algorithms that are able to treat whole genomes in acceptable times. We developed an algorithm based on an original method where an approximation of miRNA hairpins are first searched, before reconstituting the pre-miRNA structure. The approximation step allows a substantial decrease in the number of possibilities and thus the time required for searching. Our method was tested on different genomic sequences, and was compared with CID-miRNA, miRPara and VMir. It gives in almost all cases better sensitivity and selectivity. It is faster than CID-miRNA, miRPara and VMir: it takes ≈ 30 s to process a 1 MB sequence, when VMir takes 30 min, miRPara takes 20 h and CID-miRNA takes 55 h. We present here a fast ab-initio algorithm for searching for pre-miRNA precursors in genomes, called miRNAFold. miRNAFold is available at http://EvryRNA.ibisc.univ-evry.fr/.     

Sulfur-36S stable isotope labeling of amino acids for quantification (SULAQ)

We introduce a universal metabolic labeling strategy using elemental heavy 36Sulfur (36S) called 36Sulfur stable isotope labeling of amino acids for quantification (SULAQ). In the proof of principle experiment, Pseudomonas putida KT2440 was grown in defined minimal medium with sodium benzoate or sodium succinate as the sole carbon and 32S- or 36S-sodium sulfate as the sole sulfur sources. Quantification using mass spectrometry resulted in 562 proteins with 1991 unique peptides. SULAQ technology can be a valuable alternative strategy for the quantitative comparisons in MS-based proteomics approaches characterizing bacterial and other biological samples in different growth conditions.     

Immunomagnetic bead-based recovery and real time quantitative PCR (RT iq-PCR) for sensitive quantification of aflatoxin B(1)

Aflatoxin B₁ is an unavoidable natural mycotoxin that enters the food chain by contamination of food grains and feedstuffs, potentially posing carcinogenic risks to animal and human health. Immuno-PCR methods have the potential to address the need of meeting the regulatory limits by detecting trace levels of toxins present in food and animal feeds. This paper describes a real-time immuno-quantitative PCR (RT-iqPCR) assay for quantification of aflatoxin B₁ suspended in methanol:water solution that can also serve as an extraction solvent. Immuno-PCR approaches were examined including direct vs. indirect sandwich assays using monoclonal vs. polyclonal antibodies. Our best approach was obtained using monoclonal antibodies to capture aflatoxin in solution prior to immobilizing the F_c portion of the capture antibodies onto to protein G magnetic beads. This was followed by the addition of a polyclonal ‘signal antibody’ tethered with an oligonucleotide template for a subsequent PCR assay. The RT-iqPCR assay described herein leads to the sensitive detection and quantification of aflatoxin B₁ from 10 ppb down to 0.1 ppb with high correlation (r² = 0.97) and efficiency (99.5%). The approach also detected the high-dose ‘hook effect’ phenomenon (excess antigen) which was overcome by the use of dilution protocols to eliminate false negatives that may occur at levels above quantification limits of the assay. The RT-iqPCR approach discussed here is presented as a model system that could easily be adapted for aflatoxin detection in a variety of food or animal feed samples using a simple methanol:water solution as an extraction solvent.     

New tools for labeling silica in living diatoms

Silicon biomineralization is a widespread mechanism found in several kingdoms that concerns both unicellular and multicellular organisms. As a result of genomic and molecular tools, diatoms have emerged as a good model for biomineralization studies and have provided most of the current knowledge on this process. However, the number of techniques available to study its dynamics at the cellular level is still rather limited. Here, new probes were developed specifically to label the pre-existing or the newly synthesized silica frustule of several diatoms species. It is shown that the LysoTracker Yellow HCK-123, which can be used to visualize silica frustules with common filter sets, presents an enhanced signal-to-noise ratio and allows details of the frustules to be imaged without of the use of ionophores. It is also demonstrated that methoxysilane derivatives can be coupled to fluorescein-5-isothiocyanate (FITC) to preferentially label the silica components of living cells. The coupling of labeling procedures might help to address the challenging question of the process of frustule exocytosis.