msb-quantworks

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.

The steps involved in a dMS 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.
  • Fractionation – choice of fraction number dictated by project objectives, sample type, complexity and budget.
  • LC/MS/MS – Robust capillary scale chromatography enables dMS without system related bias; interfaced to state of the art Orbitrap Velos Pro or Q Exactive mass spectrometer operating at high resolution (60,000 FWHM).
  • Data analysis – Alignment of chromatographic peaks for each m/z feature, normalization, integration and statistical analysis performed using the Thermo SEIVE™ software. Optional targeted follow-on analysis of significant changes for peptide identification.
  • Report generation – PDF report and Excel. Data rationalized and presented to ensure clarity and client side ability to understand the results.

The steps involved in a GeLC/MS experiment are:

  • Sample preparation – cell or tissue lysis, plasma or serum depletion, urine and CSF sample clean-up; protein quantitation on all samples.
  • SDS-PAGE – Invitrogen Novex mini-gels, stained with colloidal coomassie.
  • Segmentation – choice of segment number dictated by project objectives, sample type, complexity and budget.
  • 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 Orbitrap Velos Pro or Q Exactive tandem mass spectrometer.
  • Data analysis – database searching with Mascot, data validation and visualization in Scaffold with quantitation and normalization based on spectral counts; secondary analysis using MaxQuant with quantitation and normalization based on integrated peak areas. 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.

Key features of a dMS experiment are:

  • High throughput.
  • Quantitation on every multiply charged feature, not reliant on database searching.

Key features of a GeLC/MS experiment are:

  • Highest number of proteins identified; lowest throughput.
  • Dual-level quantitation.
  • Quantitation on every protein identified.
  • Low sample requirements (as low as 1 µg starting material per sample).