mouse monoclonal dshb 2c2  (Developmental Studies Hybridoma Bank)

Journal: bioRxiv

Article Title: Lossless Single-Molecule Counting To Absolute Quantify Proteoforms

doi: 10.1101/2024.03.19.585761

Figure Lengend Snippet: 8-plex PICO assay detecting different proteoforms. a . Triangular detection of 4EBP1 and its phosphorylation. Note that the redundant absolute quantification of 4EBP1 phosphorylation (measured by two ABP). b . Detection of 4EBP1 protein and its T37-T46 phosphorylation using mock and dactolisib-treated MCF7 cells. After treatment 40k MCF7 cells were lysed and PICO absolute quantified. The y-axis is in molar concentration, while the right axis is proteoforms per cell, the LOD indicated. The mean number of proteoforms per cell (ppc), from left to right, are 2530000 ± 220000 ppc, 707000 ± 21000 ppc, 694000 ± 31000 ppc, 1810000.00 ± 90000 ppc, 3400 ± 2200 ppc, 10700 ± 700 ppc. Based on ANOVA (F(5, 30) = 643.0, p < 0.0001, n = 36), dactolisib significantly perturbs both protein and phosphorylation levels (both p < 0.0001, n = 12) the latter showing a significant difference in phosphorylation (27% versus 0.4%). 4EBP1 phosphorylation as detected by the two different ABPs showed no significant difference between mock (p = 0.9999, n = 12) and dactolisib-treated (p >0.9999, n = 12) MCF7 cells demonstrating concordant absolute quantitative results. c . Depictions of PICO assay used for the detection of the cytosolic protein S6K1 and p4EBP1 along with the detection of ErbB2, ErbB3, and ErbB2:ErbB3 interaction. The 8 antibodies were concurrently applied (8-plex PICO), however, detected by two different amplification primer pairs in separate dPCR reactions, S6K1, 4EBP1-phosphorylation and ErbB2, ErbB3, respectively. Note the inability of pertuzumab to bind to the interacting protein complex. d e . Couplex counts per dPCR reactions of 8-plex PICO using 0, 5000, 10000, or 20000 lysed MCF7 cells (each 6 replicates), ABPs are indicated, normalised and λ -corrected values (see methods). All antibodies were applied in parallel see (d), (e) S6K1 and p4EBP1, ErbB2 and (f) ErbB2, ErbB3 and ErbB2:ErbB3 interaction. The asterisk (*) indicates a p-value of less than 0.05. No sign indicates non-sigficant difference from zero ((e) - normality test Shapiro-Wilk test p = 0.18839, n = 91, two-sided one-sample t-test t(90) = 1.62542, p = 0.10757, n = 91; (f) - normality test Shapiro-Wilk test p = 0.11861, n = 66, two-sided one-sample t(65) = 0.21725, p = 0.82869, n = 66). f . Mock or dactolisib-treated MCF7 cells (20k cells, low ErbB2 expression) were PICO absolute qualified using the 8-plex PICO assay described above (d), all comparisons are in mock-dactolisb order. S6K1 levels were marginally, but significantly different between mock and dactolisib treatments (468,000 ± 112,000 ppc and 567,000 ± 128,000 ppc, respectively) indicating high precision, but presumably no or minimal biological significance (t(11) = 4.845, q = 0.000347, n = 24). The pronounced and expected effects of dactolisib treatment on p4EBP1 were confirmed (875,000 ± 130,000 ppc and 24,200 ± 5,700 ppc, respectively, 36-fold difference, t(10) = 20.54, q < 0.000001, n = 29). Note that the level of p4EBP1 is susceptible to variations in culturing conditions. There was an expected, known difference in ErbB3 level, confirming literature (see main text) having 3,670 ± 1,980ppc and 5,810 ± 2,640 ppc, respectively, a 1.6-fold increase, t(13) = 2.789, q = 0.006206, n = 32. ErbB2 levels were also significantly different (10,940 ± 2,160 ppc and 21,100 ± 3,400 ppc, respectively, an 1.9-fold increase, t(17) = 11.3, q < 0.000001, n = 36). ErbB2:ErbB3 interaction was significant between treatments, however, consistent between the two antibody pairs TTZ-2F12 and TTZ-Ab90, respectively, having 4,370 ± 2,520 ppc and 8,670 ± 2,400 ppc, a 2-fold increase, t(6) = 2.959, q = 0.008528, n = 19 and 5,200 ± 1,810 ppc and 12,000 ± 4,200 ppc, an 2.3-fold increase, t(8) = 4.848, q = 0.000644, n = 21. In mock-treated MCF7 cells, approximately 44 g . Mock or dactolisib-treated BT474 cells (40k cells, high ErbB2 expression) were PICO absolute qualified using the 8-plex PICO assay described above (d), all comparisons are in mock-dactolisb order. S6K1 levels were different, having 326,000 ± 95300 and 193,000 ± 60,700 ppc, t(11) = 5.91, q = 0.000034, n = 24, confirming precision, but marginal changes. p4EBP1 levels were consistent with the dactolisib treatment, 337,000 ± 125,000 and 1,190 ± 716 ppc, t(11) = 9.32, q <0.000001, n = 24 exhibiting a high sensitivity to dactolisb and shows 283-fold phosphorylation difference. ErbB2 levels, contrary to MCF7 data, show a decrease of ErbB2 level upon dactolisib treatment, 808,000 ± 177,000 and 604,000 ± 128,000 ppc, a 1.33-fold decrease, t(17) = 9.20, q < 0.000001, n = 36. While ErbB3 protein levels were highly concordant with MCF7, having 312 ± 244 and 541 ± 142 ppc, an 1.7-fold increase, (t(4) = 2.20, q = 0.023303, n = 12). Nevertheless, ErbB2:ErbB3 interaction was only detectable in mock-treated cells, having concordant, non-significantly (see below) different levels with the TTZ-2F12 and TTZ-Ab90 ABPs and was 662 ± 343 and 831 ± 311 ppc, respectively, the LOD was 200 ppc. In dactolisib-treated BT474 cells, the ErbB2:ErbB3 interaction is extremely low or absent, as evident from the data. While, in mock-treated cells, approximately 0.09% of ErbB2 interacts with ErbB3, while nearly 100% of ErbB3 engages in interaction with ErbB2, replicating its rate-limiting behaviour seen in MCF7 cells. h . Highly concordant results between TTZ-2F12 and TTZ-Ab90 ABPs detecting the ErbB2:ErbB3 interaction from left to right are t(7) = 2.588, q = 0.0728, n = 19; t(8) = 0.8282, q = 0.524873, n = 21; t(5) = 0.6921, q = 0.524873, n = 12; t(5) = 3.690, q = 0.05715, n = 12, demonstrating the self-confirming applicability of such parallel measurement.

Article Snippet: In the iBind™ Flex Western Kit (SLF2002S, Invitrogen), the nitrocellulose membrane (IB23002, Invitrogen) was immersed in 1X iBind Flex Solution (SLF2020, Invitrogen) and subsequently incubated for 3-4 hours with primary antibodies against ErbB2 (trastuzumab, Roche), ErbB3 2F12 (MA5-12675, Invitrogen), S6K1 2C2 (SAB1412617, Sigma-Aldrich), 4EBP1 554 (AHO1382, Invitrogen), 4EBP1 phosphorylation T46 (MA5-27999, Invitrogen), GAPDH (600004-1-Ig, Proteintech) and secondary anti-human-HRP (SA00001-17, Proteintech) and anti-mouse-HRP (SA00001-1, Proteintech) antibodies.

Techniques: Concentration Assay, Amplification, Expressing

Journal: bioRxiv

Article Title: Lossless Single-Molecule Counting To Absolute Quantify Proteoforms

doi: 10.1101/2024.03.19.585761

Figure Lengend Snippet: 8-plex PICO assay detecting different proteoforms. a . Triangular detection of 4EBP1 and its phosphorylation. Note that the redundant absolute quantification of 4EBP1 phosphorylation (measured by two ABP). b . Detection of 4EBP1 protein and its T37-T46 phosphorylation using mock and dactolisib-treated MCF7 cells. After treatment 40k MCF7 cells were lysed and PICO absolute quantified. The y-axis is in molar concentration, while the right axis is proteoforms per cell, the LOD indicated. The mean number of proteoforms per cell (ppc), from left to right, are 2530000 ± 220000 ppc, 707000 ± 21000 ppc, 694000 ± 31000 ppc, 1810000.00 ± 90000 ppc, 3400 ± 2200 ppc, 10700 ± 700 ppc. Based on ANOVA (F(5, 30) = 643.0, p < 0.0001, n = 36), dactolisib significantly perturbs both protein and phosphorylation levels (both p < 0.0001, n = 12) the latter showing a significant difference in phosphorylation (27% versus 0.4%). 4EBP1 phosphorylation as detected by the two different ABPs showed no significant difference between mock (p = 0.9999, n = 12) and dactolisib-treated (p >0.9999, n = 12) MCF7 cells demonstrating concordant absolute quantitative results. c . Depictions of PICO assay used for the detection of the cytosolic protein S6K1 and p4EBP1 along with the detection of ErbB2, ErbB3, and ErbB2:ErbB3 interaction. The 8 antibodies were concurrently applied (8-plex PICO), however, detected by two different amplification primer pairs in separate dPCR reactions, S6K1, 4EBP1-phosphorylation and ErbB2, ErbB3, respectively. Note the inability of pertuzumab to bind to the interacting protein complex. d e . Couplex counts per dPCR reactions of 8-plex PICO using 0, 5000, 10000, or 20000 lysed MCF7 cells (each 6 replicates), ABPs are indicated, normalised and λ -corrected values (see methods). All antibodies were applied in parallel see (d), (e) S6K1 and p4EBP1, ErbB2 and (f) ErbB2, ErbB3 and ErbB2:ErbB3 interaction. The asterisk (*) indicates a p-value of less than 0.05. No sign indicates non-sigficant difference from zero ((e) - normality test Shapiro-Wilk test p = 0.18839, n = 91, two-sided one-sample t-test t(90) = 1.62542, p = 0.10757, n = 91; (f) - normality test Shapiro-Wilk test p = 0.11861, n = 66, two-sided one-sample t(65) = 0.21725, p = 0.82869, n = 66). f . Mock or dactolisib-treated MCF7 cells (20k cells, low ErbB2 expression) were PICO absolute qualified using the 8-plex PICO assay described above (d), all comparisons are in mock-dactolisb order. S6K1 levels were marginally, but significantly different between mock and dactolisib treatments (468,000 ± 112,000 ppc and 567,000 ± 128,000 ppc, respectively) indicating high precision, but presumably no or minimal biological significance (t(11) = 4.845, q = 0.000347, n = 24). The pronounced and expected effects of dactolisib treatment on p4EBP1 were confirmed (875,000 ± 130,000 ppc and 24,200 ± 5,700 ppc, respectively, 36-fold difference, t(10) = 20.54, q < 0.000001, n = 29). Note that the level of p4EBP1 is susceptible to variations in culturing conditions. There was an expected, known difference in ErbB3 level, confirming literature (see main text) having 3,670 ± 1,980ppc and 5,810 ± 2,640 ppc, respectively, a 1.6-fold increase, t(13) = 2.789, q = 0.006206, n = 32. ErbB2 levels were also significantly different (10,940 ± 2,160 ppc and 21,100 ± 3,400 ppc, respectively, an 1.9-fold increase, t(17) = 11.3, q < 0.000001, n = 36). ErbB2:ErbB3 interaction was significant between treatments, however, consistent between the two antibody pairs TTZ-2F12 and TTZ-Ab90, respectively, having 4,370 ± 2,520 ppc and 8,670 ± 2,400 ppc, a 2-fold increase, t(6) = 2.959, q = 0.008528, n = 19 and 5,200 ± 1,810 ppc and 12,000 ± 4,200 ppc, an 2.3-fold increase, t(8) = 4.848, q = 0.000644, n = 21. In mock-treated MCF7 cells, approximately 44 g . Mock or dactolisib-treated BT474 cells (40k cells, high ErbB2 expression) were PICO absolute qualified using the 8-plex PICO assay described above (d), all comparisons are in mock-dactolisb order. S6K1 levels were different, having 326,000 ± 95300 and 193,000 ± 60,700 ppc, t(11) = 5.91, q = 0.000034, n = 24, confirming precision, but marginal changes. p4EBP1 levels were consistent with the dactolisib treatment, 337,000 ± 125,000 and 1,190 ± 716 ppc, t(11) = 9.32, q <0.000001, n = 24 exhibiting a high sensitivity to dactolisb and shows 283-fold phosphorylation difference. ErbB2 levels, contrary to MCF7 data, show a decrease of ErbB2 level upon dactolisib treatment, 808,000 ± 177,000 and 604,000 ± 128,000 ppc, a 1.33-fold decrease, t(17) = 9.20, q < 0.000001, n = 36. While ErbB3 protein levels were highly concordant with MCF7, having 312 ± 244 and 541 ± 142 ppc, an 1.7-fold increase, (t(4) = 2.20, q = 0.023303, n = 12). Nevertheless, ErbB2:ErbB3 interaction was only detectable in mock-treated cells, having concordant, non-significantly (see below) different levels with the TTZ-2F12 and TTZ-Ab90 ABPs and was 662 ± 343 and 831 ± 311 ppc, respectively, the LOD was 200 ppc. In dactolisib-treated BT474 cells, the ErbB2:ErbB3 interaction is extremely low or absent, as evident from the data. While, in mock-treated cells, approximately 0.09% of ErbB2 interacts with ErbB3, while nearly 100% of ErbB3 engages in interaction with ErbB2, replicating its rate-limiting behaviour seen in MCF7 cells. h . Highly concordant results between TTZ-2F12 and TTZ-Ab90 ABPs detecting the ErbB2:ErbB3 interaction from left to right are t(7) = 2.588, q = 0.0728, n = 19; t(8) = 0.8282, q = 0.524873, n = 21; t(5) = 0.6921, q = 0.524873, n = 12; t(5) = 3.690, q = 0.05715, n = 12, demonstrating the self-confirming applicability of such parallel measurement.

Article Snippet: Pertuzumab (PTZ, anti-ErbB2, Roche), trastuzumab (TTZ, anti-ErbB2, Roche), anti-ErbB3 2F12 (MA5-12675, Invitrogen), anti-ErbB3 Ab90 (MA5-12867, Invitrogen), penta-HIS (34660, QIAGEN), anti-TRX (4C12H10, antikoerper-online), recombinant anti-STREP II (11A7, Abcam), GAPDH (1E6D9, Proteintech), anti-4EBP1 554 (AHO1382, Invitrogen), anti-4EBP1 4F3 (H00001978-M01; Abnova), anti-4EBP1 phosphorylation T46 (MA5-27999, Invitrogen), anti-S6K1 2C2 (SAB1412617, Sigma-Aldrich), and anti-S6K1 (609902, Biolegends) antibodies were used as indicated.

Techniques: Concentration Assay, Amplification, Expressing