Figure 1.
Liquid Tissue-Selected Reaction Monitoring (SRM) workflow for quantification of proteins from formalin-fixed paraffin-embedded (FFPE) tissue.
Tissue sections are cut onto Director microdissection slides. After they are deparaffinized, areas of interest are identified by pathologists. Laser microdissection is used to procure areas of interest and the targeted cellular material is collected in a tube. The cellular material is processed using the Liquid Tissue protocol, which includes trypsinization, to produce a soluble peptide lysate. A known amount of heavy internal standard peptide is added to the lysate and the sample is analyzed using SRM to measure the absolute abundance of the endogenous peptide of interest.
Figure 2.
Development of SRM assay for Met showing the fragmentation spectrum for heavy TEFTTALQR peptide
(A), the standard curve generated in human SK-BR-3 cell lysate (B); inset: the standard curve generated without the highest spiking point (25000 amol). The total ion chromatograms for the light and heavy isotopically labeled peptides are shown (C) along with the transition ions used to identify and quantitate each peptide (D).
Table 1.
Characteristics of Met light peptide spiked SK-BR-3 lysates used to prepare calibration curve to determine limit of detection (LOD) and quantitation (LOQ) for development of Met Liquid Tissue-SRM assay.
Figure 3.
Precision assessment for measuring the absolute abundance of Met in 9 NSCLC and 11 GEC FFPE tissues.
Each sample was analyzed on two different liquid chromatography-mass spectrometry systems operated by two different scientists. Red square: Met positive GEC tumors (4/11). Blue square: Met positive NSCLC tumors (5/9).
Figure 4.
Comparison of Met levels measured in five different cell lines using Liquid Tissue-SRM and an electrochemiluminescent (ECL) immunoassay.
Inset: comparison of SRM and ECL Met levels measured within the four cell lines containing the lowest concentration of Met.
Figure 5.
Temporal reproducibility of FFPE sections processed and analyzed using Liquid Tissue-SRM over one year apart.
SD, standard deviation.
Figure 6.
Absolute levels of Met in those GEC tumors above the LOD (45/130) as measured using Liquid Tissue-SRM.
Blue indicates MET gene amplified samples (ratio of MET/CEP7 ≥2) as determined by FISH, and green indicates those not gene amplified (ratio<2). Samples in red were not FISH tested. Sample 36 (526.93 amol/ug) is taken from reference 18 (sample obtained at disease recurrence after initial onartuzumab treatment).
Figure 7.
Correlation of Met levels using Liquid Tissue-SRM and MET gene amplification by FISH in 30 GEC tumors.
This cohort includes diploid/low polyploid, high polyploid, and amplified samples. The left y-axis (blue diamond) represents the MET copy number per nucleus and the right y-axis (red square) indicates MET:CEP7 ratio.
Figure 8.
assay correlations. Correlation of IHC Met positive H-score to (A) Met SRM (amol/µg) or (B) MET/CEP7 ratio by FISH. Inset tables assess sensitivity/specificity of IHC H-score, assuming SRM (A) and FISH (B) as the comparative standards.
Table 2.
Comparison of Met IHC (≥25% or ≥50% positivity at any intensity ≥1+) with Met protein levels (by SRM) in 44 gastroesophageal cancer FFPE tissues.
Figure 9.
Three-way SRM-IHC-FISH assay correlations.
(A) Correlation of SRM Met level (amol/µg, x-axis) to Met H-Score by IHC (blue, left y-axis) and to FISH MET/CEP7 ratio (red, right y-axis) in GEC FFPE tissues having all three tests performed. Correlation coefficients for the comparisons are in their respective colors. (B) Correlation of FISH MET/CEP7 ratio (x-axis) to Met H-Score by IHC (blue, left y-axis) and to SRM Met level (red, right y-axis) in GEC FFPE tissues having all three tests performed. Correlation coefficients for the comparisons are in their respective colors.