Immuno-Parallel Reaction Monitoring (iPRM)

Immuno-PRM assay data and corresponding western blot results for phosphorylated targets in human renal carcinoma cell line ACHN under perturbation of Interleukin-4 (IL-4). The cells were treated with Human Interleukin-4 (hIL-4)#8919 at 40 ng/ml for 20 min before harvest. For iPRM assays, ACHN cell proteins were digested by trypsin, then 100 µg of ACHN tryptic peptides were mixed with heavy isotope labeled peptides, which served as internal standards. A multiplex immuno-precipitation was performed using 5 µg of each of the following CST antibodies: AKT S473 (#4060), CDK1/2/3 Y15 (#4539), PRAS40 T246 (#2997), STAT3 Y705(#9145), STAT5A/B Y694 (#9314), STAT6 Y641 (#9364), ERK1 T202/Y204(#4370), ERK2 T185(#4370), ERK2 T185 (#4370), Jun S73(#3270). Following immuno-enrichment, a multiplex PRM analysis of peptides was achieved in a Q Exactive™ mass spectrometer. Peak areas were then extracted in Skyline, and peptide concentrations were calculated based on peptide light-to-heavy ratios. Immuno-capture and PRM analysis were performed in triplicate. The same antibodies used for iPRM assays were also applied for western blotting. Twenty micrograms of protein extracts from control and treated ACHN cells were loaded on a 4% to 20% SDS-PAGE gradient gel for each blot. After transferred to nitrocellulose membrane, proteins were probed with phospho-specific antibodies at a dilution ratio of 1:1000.

Immuno-PRM assay data and corresponding western blot results for phosphorylated targets in human renal carcinoma cell line ACHN under perturbation of Interleukin-4 (IL-4). The cells were treated with Human Interleukin-4 (hIL-4)#8919 at 40 ng/ml for 20 min before harvest. For iPRM assays, ACHN cell proteins were digested by trypsin, then 100 µg of ACHN tryptic peptides were mixed with heavy isotope labeled peptides, which served as internal standards. A multiplex immuno-precipitation was performed using 5 µg of each of the following CST antibodies: AKT S473 (#4060), CDK1/2/3 Y15 (#4539), PRAS40 T246 (#2997), STAT3 Y705(#9145), STAT5A/B Y694 (#9314), STAT6 Y641 (#9364), ERK1 T202/Y204(#4370), ERK2 T185(#4370), ERK2 T185 (#4370), Jun S73(#3270). Following immuno-enrichment, a multiplex PRM analysis of peptides was achieved in a Q Exactive™ mass spectrometer. Peak areas were then extracted in Skyline, and peptide concentrations were calculated based on peptide light-to-heavy ratios. Immuno-capture and PRM analysis were performed in triplicate. The same antibodies used for iPRM assays were also applied for western blotting. Twenty micrograms of protein extracts from control and treated ACHN cells were loaded on a 4% to 20% SDS-PAGE gradient gel for each blot. After transferred to nitrocellulose membrane, proteins were probed with phospho-specific antibodies at a dilution ratio of 1:1000.

Comparison of Skyline-extracted peaks for various concentrations of phosphopeptide LAS*PELER++ from Jun (S73) after PRM (top row) and immuno-PRM (bottom row) analysis. The peaks of fragment ions (y7-98+, y6-98+, y5+, y3+, y7-98++, y6-98++) were extracted with mass tolerance 0.05 m/z from either direct injection of 1 µg trypsin digested SKBR3 lysate (top row; PRM) or from injection after immuno-precipitation of 100 µg SKBR3 peptides (bottom row; iPRM). The concentrations of the peptide in SKBR3 cell lysates were 0, 0.2, 2, 20, 200, 2000 amol/µg from left to right. The lower limit of detection (LLOD) for the peptide was enhanced from 2000 amol/µg using PRM method to 2 amol/µg using iPRM workflow.

Comparison of Skyline-extracted peaks for various concentrations of phosphopeptide LAS*PELER++ from Jun (S73) after PRM (top row) and immuno-PRM (bottom row) analysis. The peaks of fragment ions (y7-98+, y6-98+, y5+, y3+, y7-98++, y6-98++) were extracted with mass tolerance 0.05 m/z from either direct injection of 1 µg trypsin digested SKBR3 lysate (top row; PRM) or from injection after immuno-precipitation of 100 µg SKBR3 peptides (bottom row; iPRM). The concentrations of the peptide in SKBR3 cell lysates were 0, 0.2, 2, 20, 200, 2000 amol/µg from left to right. The lower limit of detection (LLOD) for the peptide was enhanced from 2000 amol/µg using PRM method to 2 amol/µg using iPRM workflow.

Immuno-PRM response curve data in linear (top) and log space (bottom) for phospho-STAT6 (Y641) peptide GY*VPATIK. Multipoint experiments were performed by immunoprecipitating increasing amounts of 10 heavy isotope-labeled synthetic peptides in a background of 100 µg of tryptic peptides derived from the breast cancer cell line SKBR-3 with light peptides spiked in as internal standards. Following immunoprecipitation, peptides were analyzed on a Q Exactive™ Mass Sepctrometer. Peptide abundance was determined by extracting fragment peptide area in Skyline. Quantitation was achieved by calculating the ratio of peak areas (heavy/light) of the 3 to 6 abundant fragment ions for each peptide. The current figures show representative data for one phosphopeptide, while a 10-target multiplex PRM assay was performed. The error bars indicate the minimum and maximum of the peak area ratios of the three capture and PRM replicates. The response curve shows a linear range of over 4 orders of magnitude and an LLOQ of 0.039 fmol/100 µg for phosphopeptide GY*VPATIK.

Immuno-PRM response curve data in linear (top) and log space (bottom) for phospho-STAT6 (Y641) peptide GY*VPATIK. Multipoint experiments were performed by immunoprecipitating increasing amounts of 10 heavy isotope-labeled synthetic peptides in a background of 100 µg of tryptic peptides derived from the breast cancer cell line SKBR-3 with light peptides spiked in as internal standards. Following immunoprecipitation, peptides were analyzed on a Q Exactive™ Mass Sepctrometer. Peptide abundance was determined by extracting fragment peptide area in Skyline. Quantitation was achieved by calculating the ratio of peak areas (heavy/light) of the 3 to 6 abundant fragment ions for each peptide. The current figures show representative data for one phosphopeptide, while a 10-target multiplex PRM assay was performed. The error bars indicate the minimum and maximum of the peak area ratios of the three capture and PRM replicates. The response curve shows a linear range of over 4 orders of magnitude and an LLOQ of 0.039 fmol/100 µg for phosphopeptide GY*VPATIK.

Immuno-PRM 5-day repeatability data for phosphopeptide IGEGTY*GVVYK from CDK1/2/3 (Y15). Samples are prepared by spiking 10 heavy isotope-labeled peptides at three levels (500 fmol, 20 fmol, 0.8 fmol as High, Med, Low level) in a background of 100 µg of tryptic peptides derived from the breast cancer cell line SKBR-3 with light peptides spiked in as internal standards. Triplicate enrichments were performed independently on 5 different days to measure repeatability. Following immunoprecipitation, peptides were analyzed on a Q Exactive™ Mass Spectrometer. Peak areas of 3 to 6 abundant fragments for each peptide were extracted in Skyline, and the peak area ratios were calculated based on the sum of fragments from light and heavy peptides. Average intra-assay and inter-assay CVs were measured for different starting concentrations of each peptide. The total CV was the square root of the sum of squares of intra- and inter-assay CVs. For phosphopeptide IGEGTY*GVVYK shown in the figure, all CVs are within 10%. For other peptides in the 10-target multiplex iPRM assay, all CVs are within 20%.

Immuno-PRM 5-day repeatability data for phosphopeptide IGEGTY*GVVYK from CDK1/2/3 (Y15). Samples are prepared by spiking 10 heavy isotope-labeled peptides at three levels (500 fmol, 20 fmol, 0.8 fmol as High, Med, Low level) in a background of 100 µg of tryptic peptides derived from the breast cancer cell line SKBR-3 with light peptides spiked in as internal standards. Triplicate enrichments were performed independently on 5 different days to measure repeatability. Following immunoprecipitation, peptides were analyzed on a Q Exactive™ Mass Spectrometer. Peak areas of 3 to 6 abundant fragments for each peptide were extracted in Skyline, and the peak area ratios were calculated based on the sum of fragments from light and heavy peptides. Average intra-assay and inter-assay CVs were measured for different starting concentrations of each peptide. The total CV was the square root of the sum of squares of intra- and inter-assay CVs. For phosphopeptide IGEGTY*GVVYK shown in the figure, all CVs are within 10%. For other peptides in the 10-target multiplex iPRM assay, all CVs are within 20%.