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Promega Corporation

Quant-Works

quant-works

The Quant-works services are designed to enable you to answer your quantitative proteomics questions. The services include workflows for label free (spectral counting and dMS), chemical labeling (iTRAQ/TMT) and metabolic labeling (SILAC). The applications of these services are numerous and include biomarker discovery and verification, studying off target effects, pharmacodynamics, cell biology and general purpose differential analysis.

Label Free

This service enables the quantitative and qualitative analysis of complex samples such as cell lysates, tissue homogenates or biofluids. The results from this experiment provide a catalogue of the proteins present in all samples and a statistical analysis reflecting the changes in the protein levels observed across the samples, along with associated pathway information.

Differential mass spectrometry (dMS) is a workflow that provides relative quantitation and identifies statistically significant changes in full scan mass spectrometry data. dMS requires no metabolic or chemical labeling, or sample pooling. Data from one sample can be compared to data from many others. In a dMS experiment LC/MS data are acquired using the full scan mode of operation. Post-acquisition data processing is used to collate a ranked list of differences between samples from multiple conditions.

GeLC/MS technique uses an offline first dimension SDS-PAGE protein level separation to reduce the complexity of samples prior to an online second dimension reverse phase peptide level separation. The protein sample resolved on the SDS gel is divided into a number of segments which are digested with trypsin prior to the online analysis. The number of segments dictates the depth of coverage and the time to analyze a single sample. Digested samples are analyzed by LC/MS/MS and protein identification data are collated into a non-redundant list per gel lane. For differential experiments quantitation is performed following normalization using spectral counting and /or Normalized Spectral Abundance Factor values.

SERVICE ID SERVICE DESCRIPTION LIST PRICE PER SAMPLE
MSB-15 Label Free, unfractionated $775
MSB-16 Label Free, 10 fractions $1,450
MSB-17 Label Free, 20 fractions $2,100
MSB-18 Label Free, 40 fractions $3,500

ITRAQ and TMT

Chemical labeling methods such as iTRAQ and TMT enable increased throughput in quantitative proteomics experiments. Samples are digested with trypsin and are subsequently labeled with isobaric tagging reagents, tagged samples are then pooled prior to analysis. The tag chemistry is such that tagged peptides from different samples in a pool will have the same precursor ion m/z and under collision induced dissociation conditions will yield diagnostic tag specific reporter ions that can be used for quantitation. Thus a peptide fragmentation spectrum contains not only information on the identity of the peptide but also information on the relative amounts of that peptide in each pooled sample. Data processing software is used to interpret the peptide level data and report quantitative results for detected proteins.

To improve the sensitivity of the chemical labeling techniques we use off-line strong cation exchange (SCX) chromatography to fractionate the pooled sample prior to LC/MS/MS analysis.

The steps involved in a chemical labeling experiment are:

  • Sample preparation – cell or tissue lysis, plasma or serum depletion, urine and CSF sample clean-up; protein quantitation on all samples.
  • Sample digestion – reduction and alkylation followed by in-solution trypsin digestion.
  • Sample labeling – Reagents are available that allow the multiplexing of 2, 4, 6 or 8 samples.
  • Sample pooling – equal amounts of labeled samples are combined.
  • Fractionation – fractionation of pooled sample using off-line SCX.
  • Nano LC/MS/MS – 250 nL/min liquid chromatography to yield high sensitivity; interfaced to a state of the art Velos Orbitrap Pro tandem mass spectrometer.
  • Data analysis – database searching with Mascot, data validation, visualization and quantitation using Scaffold Q+.
  • Report generation – PDF report and Excel. Data rationalized and presented to ensure clarity and the ability to understand the results.
SERVICE ID SERVICE DESCRIPTION LIST PRICE PER SAMPLE
MSB-19 iTRAQ $9,200
MSB-20 TMT $7,875

SILAC

This service is designed for the quantitative and qualitative analysis of cell based systems. The results from this experiment provide a catalogue of the proteins present in all samples and a statistical analysis reflecting the changes in the protein levels observed across the samples, along with associated pathway information.

Stable isotope labeling with amino acids in cell culture (SILAC) is a quantitative proteomics technique that involves the incorporation of a label into proteins in vitro prior to analysis by LC/MS/MS. Cells are labeled during the culturing process using media containing light or heavy amino acids, the heavy amino acids have stable isotope atoms incorporated e.g. lysine (13C6H1215N2O) or arginine (13C6H1215N4O2). The labeled amino acids are used in protein synthesis, after several passages the labeled residues have been fully incorporated into the proteins. The labeled amino acids are equivalent to their unlabeled counterparts and their presence has no impact on the biological system.

Cells from light and heavy cultures can be lysed and mixed to create a population of both heavy and light labeled pairs of proteins. The pooled sample is then processed using our GeLC/MS approach. The GeLC/MS technique uses an offline first dimension SDS-PAGE protein level separation to reduce the complexity of samples prior to an online second dimension reverse phase peptide level separation. The protein sample resolved on the SDS gel is divided into a number of segments which are digested with trypsin prior to the online analysis. The number of segments dictates the depth of coverage and the time to analyze a single sample. Digested samples are analyzed by LC/MS/MS and protein identification data are collated into a non-redundant list per gel lane. Protein differential expression is calculated as a function of the peak areas of the light and heavy peptides detected in the sample.

The steps involved in a chemical labeling experiment are:

  • Sample preparation – cell lysis.
  • Label incorporation analysis – a screen to ensure full incorporation of labeled amino acids into the system. Data are evaluated with MaxQuant.
  • SDS-PAGE – Invitrogen Novex mini-gels, stained with colloidal coomassie.
  • Segmentation – 10, 20 or 40 segments. The number of segments is dictated by project objectives, sample type, complexity and budget. The default number of segments for this service is 40.
  • Sample digestion – robotic reduction and alkylation followed by trypsin digestion.
  • Nano LC/MS/MS – 250 nL/min liquid chromatography to yield high sensitivity; interfaced to state-of-the-art Velos Orbitrap tandem mass spectrometer.
  • Data analysis – analysis using MaxQuant with quantitation and normalization based on integrated peak areas. Protein identification is performed with Mascot. Statistical assessment of significant differences and optional pathway analysis.
  • Report generation – PDF report, Excel and Scaffold files. Data rationalized and presented to ensure clarity and the ability to understand the results.
SERVICE ID SERVICE DESCRIPTION LIST PRICE PER SAMPLE
MSB-21 SILAC, 40 fractions $4,100

SRM/MRM

Liquid Chromatography-Selected Reaction Monitoring/Mass Spectrometry (LC-SRM/MS) is a technique commonly associated with the selective and sensitive detection of small molecules (<1000Da). The technique is commonplace in the clinical, pharmaceutical and environmental monitoring fields and is recognized for enabling robust, high throughput assays. LC-SRM/MS is an emerging technology for the quantitation of proteins. The principle underlying this approach is one of surrogacy; a proteolytic peptide produced from the enzymatic digestion of a protein can be monitored and quantified using LC-SRM/MS. The calculated molar amount of the surrogate peptide can be used to infer the concentration of the protein, assuming a 1:1 mole ratio between the peptide and the protein. This method offers an opportunity to dramatically reduce assay development times for proteins of interest and has all of the positive attributes associated with small molecule applications.

This service is designed to enable access to this emerging technology for use on your own biological systems. This service involves the quantitation of ten proteins in ten samples using single point calibration. Internal standard peptides are not included in the service but MS Bioworks is happy to assist in sourcing and purchasing suitable reagents.

The steps involved in a Selected Reaction Monitoring experiment are:

  • Method development – peptide selection via a combined in-silico and empirical approach using Skyline software. We aim to select five surrogate peptides for each protein in the assay but this number is protein specific.
  • Sample digestion – robotic reduction and alkylation followed by trypsin digestion.
  • Optional – addition of synthetic peptide standards.
  • LC/MS/MS – robust liquid chromatography to yield high sensitivity; interfaced to a TSQ Quantum Ultra to allow sensitive time scheduled data acquisition.
  • Data analysis – data are analyzed using Skyline software.
  • Report generation – PDF report, Excel and Scaffold files. Data are rationalized and presented to ensure clarity and the ability to understand the results.
SERVICE ID SERVICE DESCRIPTION LIST PRICE PER SAMPLE
MSB-22 SRM/MRM $9,500

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