Near-infrared fluorescence detection of ATP-biotin-mediated phosphoprotein labeling

Detection of phosphoproteins plays an important role in understanding protein function in cellular signalling pathways. Improved methods for identification and quantification of phosphoproteins are research priorities. Near-infrared (NIR) fluorescence detection of a γ-modified ATP-biotin analog was used to detect protein phosphorylation, using both model kinase substrates and mammalian cell lysates. NIR signal intensity was dependent on substrate and ATP-biotin concentrations.     

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta.

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.     

Rapid two-stage polymerase chain reaction amplification of chromosomal DNA segments in lysates made from monolayer cultures attached to microcarrier beads

We describe a method for the rapid two-stage amplification and detection by ethidium bromide staining of chromosomal nucleotide (nt) sequences in lysates made directly from anchorage-dependent cells attached to microcarrier beads. The procedure circumvents the need for cell detachment steps prior to analysis, facilitates the collection, transfer, and manipulation of the cells being studied, and makes unnecessary the use of Southern-blot hybridization for identification of specific nt sequences present in a small fraction of cells within a heterogeneous population.     

Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag-based reagents

Recent development of the phosphate chelator, Phos-tag, together with Phos-tag pendant reagents, has provided new methods for detection of phosphorylated serine, threonine, tyrosine, and histidine residues in phosphoproteins. We have investigated the use of Phos-tag for detection and quantification of phospho-aspartate in response regulator proteins that function within two-component signaling systems. Alternative methods are especially important, because the labile nature of the acylphosphate bond in response regulator proteins has restricted the application of many traditional methods of phosphoprotein analysis. We demonstrate that Phos-tag gel stain can be used to detect phospho-Asp in response regulators and that Phos-tag acrylamide gel electrophoresis can be used to separate phosphorylated and unphosphorylated forms of response regulator proteins. The latter method, coupled to Western blot analysis, enables detection of specific phosphorylated proteins in complex mixtures such as cell lysates. Standards of phosphorylated proteins can be used to correct for hydrolysis of the labile phospho-Asp bond that invariably occurs during analysis. We have employed Phos-tag methods to characterize the phosphorylation state of the Escherichia coli response regulator PhoB both in vitro, using purified protein, and in vivo, by analyzing lysates of cells grown under different conditions of induction of the PhoR/PhoB phosphate assimilation pathway.     

A Rapid Screening Assay to Search for Phosphorylated Proteins in Tissue Extracts

Reversible protein phosphorylation is an essential mechanism in the regulation of diverse biological processes, nonetheless is frequently altered in disease. As most phosphoproteome studies are based on optimized in-vitro cell culture studies new methods are in need to improve de novo identification and characterization of phosphoproteins in extracts from tissues. Here, we describe a rapid and reliable method for the detection of phosphoproteins in tissue extract based on an experimental strategy that employs 1D and 2D SDS PAGE, Western immunoblotting of phosphoproteins, in-gel protease digestion and enrichment of phosphorpeptides using metal oxide affinity chromatography (MOAC). Subsequently, phosphoproteins are identified by MALDI-TOF-MS/MS with the CHCA-TL or DHB ML sample matrix preparation method and further characterized by various bioinformatic software tools to search for candidate kinases and phosphorylation-dependent binding motifs. The method was applied to mouse lung tissue extracts and resulted in an identification of 160 unique phosphoproteins. Notably, TiO₂ enrichment of pulmonary protein extracts resulted in an identification of additional 17 phosphoproteins and 20 phosphorylation sites. By use of MOAC, new phosphorylation sites were identified as evidenced for the advanced glycosylation end product-specific receptor. So far this protein was unknown to be phosphorylated in lung tissue of mice. Overall the developed methodology allowed efficient and rapid screening of phosphorylated proteins and can be employed as a general experimental strategy for an identification of phosphoproteins in tissue extracts.     

Immunoprecipitation combined with microchip capillary gel electrophoresis: Detection and quantification of β-galactosidase from crude E. coli cell lysate

A sensitive and selective analytical method for the determination and quantification of endogenous β-galactosidase in crude E. coli cell lysates by immunoprecipitation combined with automated microchip capillary gel electrophoresis (IP-MCGE) with laser-induced fluorescence (LIF) detection was developed. Total cell lysates were derivatized minimally with a fluorescence dye, incubated with anti-β-galactosidase antibodies, and the antigen/antibody complex was precipitated with protein G-coated magnetic beads. After capturing the complex, it was eluted from the beads under denaturing conditions and loaded directly onto a multisample microchip for analysis. The effects of antibody selection and the importance of preclearing steps were studied in detail. For quantification, an external calibration through spiking pure β-galactosidase into E. coli lysate was performed. Recovery rates of immunoprecipitation after spiking experiments and the amount of unknown endogenous β-galactosidase in E. coli lysates were determined. As proof of principle, E. coli cultures grown on nutrition media with several glucose/lactose ratios were analyzed. Differences in the expression level of β-galactosidase could be detected and measured with the developed method. Detected amounts of β-galactosidase in different culture media correlated with the β-galactosidase activities in these cultures.     

Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis

Prefractionation procedures facilitate the identification of lower-abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one-tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal-regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two-dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK-activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research.     

Immunochemical analysis of molecular forms of protein synthesis initiation factors in crude cell lysates of Escherichia coli

Three initiation factors (IF1, IF2, and IF3) have been highly purified from Escherichia coli and extensively characterized, but little is known about the molecular forms of these proteins as they occur in vivo. We have analyzed molecular-weight and charge forms in crude cell lysates by polyacrylamide gel electrophoresis followed by immunoblotting with antibodies specific for the initiation factors. Freshly grown bacterial cells were lysed by sonication in buffer containing sodium dodecyl sulfate, and the lysate was fractionated by gel electrophoresis. Proteins from the gel were electrotransferred to a nitrocellulose sheet which was treated with a specific rabbit antiserum followed by radiolabeled Staphylococcus aureus protein A. Autoradiography showed only one major band each for IF1 and IF3, exactly corresponding to the isolated factors. For IF2, two molecular-weight forms were detected which were identical with purified IF2a and IF2b. No evidence for precursor forms was found. Two-dimensional gel analysis showed no charge heterogeneity for IF1, IF2a, and IF3, but multiple forms were seen for IF2b. Analysis of phosphoproteins from cells grown in radioactive phosphate medium ruled out the possibility that phosphorylation occurs on the initiation factors, elongation factors, or ribosomal proteins.     

Analysis and isolation of human transferrin receptor using the OKT-9 monoclonal antibody covalently crosslinked to magnetic beads.

A method is described for the use of magnetic beads as a solid phase for the immunoprecipitation of labeled proteins. The anti-human transferrin receptor monoclonal antibody OKT-9 has been coupled to sheep anti-mouse IgG1-coated magnetic beads using the crosslinking agent dimethyl pimelimidate. The transferrin receptor is readily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography following immunoprecipitation from 35S-labeled cell lysates. When compared with precipitations using OKT-9 coupled to protein G Sepharose the magnetic beads result in fewer nonspecific bands. The protocol described is generally applicable to the identification of labeled proteins. In addition, because magnetic beads are amenable to covalent crosslinking procedures they can be used for the purification of proteins from complex mixtures. Covalently crosslinked OKT-9 sheep anti-mouse IgG1-coated magnetic beads have been used to affinity purify unlabeled transferrin receptor from cell lysates giving comparable purity and yield to transferrin Sepharose isolated transferrin receptor. The major advantages offered by magnetic beads compared to conventional affinity matrices are low nonspecific binding and the rapidity with which the purification can be performed.     

Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule

We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate-binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, we tried to determine their phosphorylation sites by MS/MS analysis after in-gel digestion of the corresponding spots on the 2DE gel to the rapid on-membrane identifications. As one example of use of information gained from the rapid-profiling approach, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3.     

Proteomic and phosphoproteomic landscape of salivary extracellular vesicles to assess OSCC therapeutical outcomes

Circulating extracellular vesicles (EVs) have emerged as an appealing source for surrogates to evaluate the disease status. Herein, we present a novel proteomic strategy to identify proteins and phosphoproteins from salivary EVs to distinguish oral squamous cell carcinoma (OSCC) patients from healthy individuals and explore the feasibility to evaluate therapeutical outcomes. Bi-functionalized magnetic beads (BiMBs) with Ti (IV) ions and a lipid analog, 1,2-Distearoyl-3-sn-glycerophosphoethanolamine (DSPE) are developed to efficiently isolate EVs from small volume of saliva. In the discovery stage, label-free proteomics and phosphoproteomics quantification showed 315 upregulated proteins and 132 upregulated phosphoproteins in OSCC patients among more than 2500 EV proteins and 1000 EV phosphoproteins, respectively. We further applied targeted proteomics by coupling parallel reaction monitoring with parallel accumulation-serial fragmentation (prm-PASEF) to measure panels of proteins and phosphoproteins from salivary EVs collected before and after surgical resection. A panel of three total proteins and three phosphoproteins, most of which have previously been associated with OSCC and other cancer types, show sensitive response to the therapy in individual patients. Our study presents a novel strategy to the discovery of effective biomarkers for non-invasive assessment of OSCC surgical outcomes with small amount of saliva.     

Identification and preliminary characterization of two related proliferation-associated nuclear phosphoproteins

Two nuclear phosphoproteins, pp35 and pp32, were purified from A20 cells, a murine B-lymphoblastoid cell line. Initially detected by cross-reactivity with antibodies to human erythrocyte protein 4.1, the 35- and 32-kDa proteins were purified by sequential fractionation of non-ionic detergent cell lysates on DEAE-cellulose, high performance liquid chromatography (HPLC)-anion-exchange chromatography, and HPLC hydroxylapatite chromatography. By two-dimensional peptide mapping, pp35 and pp32 are related but do not appear to represent sequential proteolytic products. Both pp35 and pp32 appear to be associated with cell proliferation. Antibodies specific for pp35 and pp32 show prominent intranuclear staining in A20 cells but only focal staining in normal murine lymphoid tissues. Quantitative immunoblotting showed that both pp35 and pp32 are, respectively, expressed at 5.9 x 10(4) and 7.0 x 10(4) copies/cell in small, dense resting B lymphocytes, increasing approximately 12- and 7-fold after polyclonal stimulation with lipopolysaccharide. When normalized to total cell protein, this represents specific inductions of approximately 4- and 2-fold. Expression of both pp35 and pp32 is constitutively high in populations of neoplastic B cell lines; moreover, both are expressed in the nuclei of intestinal crypt epithelial cells but not in other epithelial compartments in the same sections, suggesting that forms of pp35 and pp32 may be expressed in additional tissues and associated with proliferation.     

Reduced tyrosine phosphorylation and nonresponsiveness to EGF-mediated cytotoxicity in EGF receptor-hyperproducing UCVA-1 cells

UCVA-1 cells, derived from human pancreas adenocarcinoma, have a high number of epidermal growth factor (EGF) receptors (1.0 x 10(6) per cell) but their growth is not inhibited by EGF, unlike other EGF receptor-hyperproducing tumour cells. In UCVA-1 cells EGF activates neither the phosphatidylinositol turnover nor protein kinase C. EGF, however, enhances the phosphorylation of EGF receptors at specific tyrosine residues, indicating that the EGF receptor kinase is active and subject to autophosphorylation. Downmodulation of EGF receptors by 12-O-tetradecanoylphorbol 13-acetate (TPA) is also observed. Using an anti-phosphotyrosine antibody several phosphoproteins, including EGF receptors, were immunoprecipitated from UCVA-1 cell lysates, whereas more than 20 phosphoproteins were detected in other EGF receptor-hyperproducing tumour cells (NA), indicating that tyrosine-phosphorylation of endogenous substrates by EGF receptor kinase is significantly reduced in UCVA-1 cells. Thus, non-responsiveness of UCVA-1 cells to EGF is correlated with the reduced tyrosine phosphorylation.     

Analysis of protein phosphorylation patterns reveals unanticipated complexity in T lymphocyte activation pathways.

Protein kinases are considered likely to play important roles in the still dimly understood process by which mitogens induce resting T lymphocytes to enter the cell cycle. Using two-dimensional electrophoretic analysis of lysates from orthophosphate-labeled cells, we have compared patterns of phosphorylation in freshly isolated murine splenic T cells exposed to three mitogenic agents: antibody to the epsilon-chain of the TCR CD3 complex, the plant lectin Con A, and a mixture of PMA and ionomycin, which together bypass the signal transduction apparatus to activate intracellular pathways. Of 14 phosphoproteins found whose level of phosphorylation was increased (at least fivefold) by anti-CD3 epsilon antibody, 13 also responded to the mixture of PMA and ionomycin. Surprisingly, however, only 5 of these 14 also responded strongly to Con A exposure. We also identified two substrates that were phosphorylated in response to Con A but not to anti-CD3. Phosphorylation patterns were also studied in T cells exposed to either PMA or ionomycin alone, to gain further insight into the role of protein kinase C and calcium-dependent events in the activation process. Of 16 phosphoproteins that responded to mixtures of PMA and ionomycin, 4 were shown to require the ionomycin signal, 2 to require the PMA signal, and 3 others to respond only when both activators were present; the other 7 responded to either agonist added alone. In addition, we found two PMA-sensitive phosphoproteins in which phosphorylation was inhibited by ionomycin induced calcium signals. Finally, we identified several phosphoproteins which show differential responsiveness in CD4+ and CD8+ T cells. Classification of kinase substrates based on their differential susceptibility to these stimuli should provide new insights into the mode of action of agents and diseases that affect T cell activation.     

Highly sensitive detection of protein phosphorylation by using improved Phos-tag Biotin

We have previously shown that the dinuclear zinc(II) complex Phos-tag and its derivatives act as phosphate-capture molecules in aqueous solution under conditions of neutral pH. In this study, our aim was to develop more-advanced applications for the detection of phosphopeptides and phosphoproteins by using several newly synthesized Phos-tag derivatives, including a bisbiotinylated Phos-tag (BTL-108), a tetrakisbiotinylated Phos-tag (BTL-109), and a monobiotinylated Phos-tag with a dodeca(ethylene glycol) spacer (BTL-111), as well as the commercially available product BTL-104. Among these complexes, BTL-111 showed the best performance in Western blotting by an ECL system using HRP conjugated streptavidin. In addition, in a quartz-crystal microbalance analysis of a phosphoprotein, the presence of the long hydrophilic dodeca(ethylene glycol) spacer in a novel Phos-tag sensor chip coated with BTL-111 resulted in a greater sensitivity than was achieved with a similar chip coated with BTL-104. Moreover, a peptide microarray technique using the ECL system and BTL-111 permitted high-throughput assays for the specific and highly sensitive detection of protein kinase activities in cell lysates.     

Directed covalent immobilization of fluorescently labeled cytokines

Cytokines are important mediators coordinating inflammation and wound healing in response to tissue damage and infection. Therefore, immobilization of cytokines on the surface of biomaterials is a promising approach to improve biocompatibility. Soluble cytokines signal through receptors on the cell surface leading to cell differentiation, proliferation, or other effector functions. Random immobilization of cytokines on surfaces will result in a large fraction of inactive protein due to impaired cytokine--receptor interaction. We developed a strategy that combined (i) directed covalent coupling of cytokines, (ii) quantification of coupling efficiency through fluorescence detection, and (iii) a reliable protease cleavage assay to control orientation of coupling. For this purpose, fusion proteins of the SNAP-tag followed by an enterokinase recognition site, yellow fluorescent protein (YFP), and the cytokine of interest being either interleukin-6 (IL-6) or oncostatin M (OSM) were generated. The SNAP-tag is a derivative of O(6)-alkylguanine-DNA alkyltransferase that couples itself covalently to benzylguanine. Bioactivities of the SNAP-YFP-cytokines were shown to be comparable with the nontagged cytokines. Efficient coupling of SNAP-YFP-cytokines to benzylguanine-modified beads was demonstrated by flow cytometry. The fact that enterokinase treatment released most of the fluorescence from the beads is indicative for directed coupling and only marginal adsorptive binding. Cellular responses to SNAP-YFP-cytokine beads were analyzed in cellular lysates and by confocal microscopy indicating that the directionally immobilized cytokines are fully signaling competent with respect to the activation of ERK and STAT3. The strategy presented here is generally applicable for the directed covalent immobilization of fluorescently labeled proteins including the convenient and reliable control of coupling efficiency and orientation.     

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 °C instead of the conventional 70-100 °C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other ''large'' proteins like BRCA2 should be readily achievable.     

Identification of novel target proteins of cyclic GMP signaling pathways using chemical proteomics

For deciphering the cyclic guanosine monophosphate (cGMP) signaling pathway, we employed chemical proteomics to identify the novel target molecules of cGMP. We used cGMP that was immobilized onto agarose beads with linkers directed at three different positions of cGMP. We performed a pull-down assay using the beads as baits on tissue lysates and identified 9 proteins by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry. Some of the identified proteins were previously known cGMP targets, including cGMP-dependent protein kinase and cGMP-stimulated phosphodiesterase. Surprisingly, some of the coprecipitated proteins were never formerly reported to associate with the cGMP signaling pathway. The competition binding assays showed that the interactions are not by nonspecific binding to either the linker or bead itself, but by specific binding to cGMP. Furthermore, we observed that the interactions are highly specific to cGMP against other nucleotides, such as cyclic adenosine monophosphate (cAMP) and 5\'-GMP, which are structurally similar to cGMP. As one of the identified targets, MAPK1 was confirmed by immunoblotting with an anti-MAPK1 antibody. For further proof, we observed that the membrane-permeable cGMP (8-bromo cyclic GMP) stimulated mitogen-activated protein kinase 1 signaling in the treated cells. Our present study suggests that chemical proteomics can be a very useful and powerful technique for identifying the target proteins of small bioactive molecules.     

Protein synthesis eukaryotic initiation factors 4A and 4B are not altered by poliovirus infection of HeLa cells

Infection of HeLa cells by poliovirus results in the inhibition of translation of capped cellular mRNA. A plausible mechanism for this inhibition is that the structure of one or more initiation factors involved in the recognition of capped mRNA is altered. Eukaryotic initiation factor (eIF) 4A and eIF-4B are implicated in mRNA binding to 40 S ribosomal subunits and can be cross-linked to oxidized capped mRNA. We examined these factors in HeLa cell lysates by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. No alterations in the number of molecules/cell, in the molecular size, or in extents of covalent modification were detected when lysates from infected and mock-infected cells were compared. The integrity of eIF-2 and several eIF-3 polypeptides was also examined and likewise no alterations were detected. The failure of the translational machinery to recognize capped mRNA therefore is not due to a change in the structure of these initiation factors.     

Comparison of gel‐based phosphoproteomic approaches to analyse scarce oviductal epithelial cell samples

The reversible change of the phosphorylation state of proteins regulates key cellular processes. In the present study, three different gel‐based approaches were compared with regard to their applicability to quantitatively analyse the phosphoproteome of scarce biological material obtained ex vivo. Our results show that the phosphoproteome characterisation of oviductal epithelial cells isolated from the female reproductive tract requires affinity enrichment and pre‐electrophoretic labelling using fluorescence dyes. Using this approach, 30 μg of enriched phosphoproteins proved to be sufficient for the phosphoproteome characterisation. In contrast, sequential fluorescence staining of 2D‐separated total cell lysates as well as sequential staining in conjunction with a pre‐enrichment step led to detection discrepancies and excluded further analysis steps. Information gained from this study provides a successful approach for the phosphoproteome analysis of scarce samples. In addition, the cellular processes taking place in the female reproductive tract can be monitored ex vivo.