Multiplexed Imaging of Protein Phosphorylation on Membranes Based on Ti^IV Functionalized Nanopolymers

Abstract

Accurate protein phosphorylation analysis reveals dynamic cellular signaling events not evident from protein expression levels. The most dominant biochemical assay, western blotting, suffers from the inadequate availability and poor quality of phospho-specific antibodies for phosphorylated proteins. Furthermore, multiplexed assays based on antibodies are limited by steric interference between the antibodies. Here we introduce a multifunctionalized nanopolymer for the universal detection of phosphoproteins that, in combination with regular antibodies, allows multiplexed imaging and accurate determination of protein phosphorylation on membranes.

Sensitive Approaches for the Assay of the Global Protein Tyrosine Phosphorylation in Complex Samples Using a Mutated SH2 Domain

“…HeLa cell lysates were obtained from the cells that were untreated or treated with EGF for 20 min, and then the same amounts of proteins were separated by the SDS-PAGE, transferred onto a membrane, and blotted independently using Trm-SH2, Wt-SH2, or P-Tyr-100, or by directly using GST polyclonal antibody as the control. It can be seen from Figure that a significant increase in signal was observed for all three approaches, which was consistent with the fact that the tyrosine phosphorylation level increases significantly after EGF stimulation. − The above results indicate that these methods were all responsive to the tyrosine phosphorylation level. However, the patterns were quite different.…”

“…However, such a method is seldom reported due to a lack of pan-specific reagents recognizing the phosphorylation sites. Recently, Tao et al presented an interesting approach to measure the total protein phosphorylation level using a reagent termed pIMAGO that is multifunctionalized with titanium ions for its superior selectivity toward phosphorylated sites. ,, Tyrosine phosphorylation plays an important role for regulating biological processes. However, Tao’s method was unable to differentiate the phosphorylation that occurred on Ser, Thr, or Tyr and therefore cannot be used to determine the total tyrosine phosphorylation level.…”

“…EGF signaling begins with activation of the EGF receptor by tyrosine phosphorylation and extends through a cascade of downstream kinases to mediate the phosphorylation of a large number of substrate proteins . Several MS studies have focused on the global tyrosine phosphorylated “early events” after growth-factor stimulation. ,,, These approaches employed the immunoprecipitation by antiphosphotyrosine antibodies, and large numbers of tyrosine phosphorylation sites were identified and quantified as a function of stimulus, time, and subcellular location, providing valuable dynamic information on tyrosine phosphorylated proteins and closely associated binders. While it needs large amounts of proteins, and more importantly, it is often limited to analysis of the dynamical processes with less than 3 time points due to the high cost and low throughput of the approach.…”

“…The activation of the EGFR signal pathway was verified by the antibodies of p44/42 MAPK (Erk1/2) (137F5), phospho-p44/42 MAPK (Erk1/2) (Figure S3a), and phospho-EGF receptor antibody sampler kit (Figure S3b). ,, The obtained cell lysates corresponding to 200 ng of proteins were added to different wells and detected with Trm-SH2. As illustrated in Figure b, a significant increase in signal was observed when the cells were treated with EGF for 1 min, and then the signal decayed afterward.…”

Phosphorylation of Extracellular Bone Matrix Proteins and Its Contribution to Bone Fragility

“…These techniques laid an invaluable foundation for understanding protein phosphorylation and are still very useful, but typically the approaches used focus on a small number of individual proteins. Recent technological advances have opened new avenues for studies of protein phosphorylation events on a more global and systematic scale …”