Site‐specific separation and detection of phosphopeptide isomers with pH‐mediated stacking capillary electrophoresis‐electrospray ionization‐tandem mass spectrometry

This study reported a pH‐mediated stacking CE coupled with ESI MS/MS method to determine the phosphorylation sites of three synthetic phosphopeptides containing structural isomers. These phosphopeptides mimic the phosphopeptides (amino acid residues 12–25) derived from the trypsin‐digested products of human lamin A/C protein. The LODs were determined to be 118, 132 and 1240 fmol for SGAQASS¹⁹TpPL²²SPTR, SGAQASS¹⁹TPL²²SpPTR, and SGAQASS¹⁹TpPL²²SpPTR, respectively. The established method was employed to analyze the phosphorylation sites of the trypsin‐digested products of glutathione S‐transferase‐lamin A/C (1–57) fusion protein that had been phosphorylated in vitro by cyclin‐dependent kinase 1. The results indicated that this method is feasible to specifically determine the phosphorylation site from phosphopeptide isomers in the trypsin‐digested products of a kinase‐catalyzed phosphoprotein, which should benefit the investigation of protein kinase‐mediated cellular signal transduction.     

Probing the role of the linker in ferrocene-ATP conjugates: monitoring protein kinase catalyzed phosphorylations electrochemically

The synthesis and electrochemical properties of ferrocene conjugates are presented for the purpose of investigating adenosine 5′‐[γ‐ferrocenoylalkyl] triphosphate ( 1 a – 4 a , ferrocene (Fc)–ATP) as co‐substrates for phosphorylation reactions. Compounds 1 a – 4 a were synthesized, purified by HPLC, and characterized by NMR spectroscopy and mass spectrometry. In solution, all Fc–ATP bioconjugates exhibit a reversible one‐electron redox process with a half‐wave potential (E_1/2) in the 390–430 mV range, peak separations (ΔE_p) in the 40–70 mV range, and the peak current ratio (i_pa/i_pc) near unity. The peptide‐modified surface Glu‐Gly‐Ile‐Tyr‐Asp‐Val‐Pro was used to study the sarcoma‐related protein (Src) kinase activity by employing the Fc–ATP bioconjugates as co‐substrates. Subsequent kinase‐catalyzed transfer of the γ‐Fc‐phosphate group to the tyrosine residues of the surface‐bound peptides was characterized by a formal potential (E^o) ≈390 mV (vs. Ag/AgCl). The Fc‐coverage, estimated by time‐of‐flight secondary‐ion mass spectrometry (TOF‐SIMS) and cyclic voltammetry (CV), suggested validity of Fc–ATP conjugates as kinase co‐substrates. Depending on the length of the alkyl spacer of the Fc–ATP conjugate, different current densities were obtained, pointing to a direct correlation between the two. Molecular modeling revealed that the structural constraint imposed by the short alkyl spacer ( 1 a ) causes a steric congestion and negatively affects the outcome of phosphorylation reaction. An optimal analytical response was obtained with the Fc–ATP conjugates with linker lengths longer than six CH₂ groups.     

Analysis of protein phosphorylation combining capillary electrophoresis with ATP analog labeling technique

Protein phosphorylation is one of the most basic mechanisms for regulating and controlling protein biological activity and function, and it is also a very important posttranslational modification process. Protein phosphorylation participates in and regulates many life activities such as signal transduction, gene expression, cell cycle, and so on. In this paper, we propose a method for the determination of the protein phosphorylation combining capillary electrophoresis (CE) with ATP analog labeling technique. We synthesized two new ATP analogs (ATP-NB and ATP-TATD-NB) functionalized by norbornene. Using Abl kinase as a model, we established a method for the determination of the kinase activity in solution and lysate by CE with laser-induced fluorescence detection (CE-LIF). This method was used to evaluate the efficiencies of kinase inhibitors. The IC₅₀ values obtained are basically consistent with the reports. By D–A reaction (inverse electron demand Diels–Alder reaction) to label TZ-BODIPY fluorescence, we also realized the phosphorylation fluorescence detection of substrate peptide. Then, we used fluorescence confocal microscopy imaging technology to study the phosphorylation of proteins in vivo by the D–A reaction of ATP-NB and TZ-BODIPY. Our preliminary results documented that the combination of CE-LIF with analog ATP-NB labeling technique is an effective strategy for the determination of the protein phosphorylation and the kinase activity and for screening of kinase inhibitors. The D–A reaction of ATP-NB and TZ-BODIPY also laid the foundation for the subsequent in situ study of protein phosphorylation.     

Proteomic identification of p38 MAP kinase substrates using in vitro phosphorylation

Protein phosphorylation is a major mechanism that regulates many basic cellular processes. Identification and characterization of substrates for a given protein kinase can lead to a better understanding of signal transduction pathways. However, it is still difficult to efficiently identify substrates for protein kinases. Here, we propose an integrated proteomic approach consisting of in vitro dephosphorylation and phosphorylation, phosphoprotein enrichment, and 2D‐DIGE. Phosphatase treatment significantly reduced the complexity of the phosphoproteome, which enabled us to efficiently identify the substrates. We employed p38 mitogen‐activated protein kinase (p38 MAP kinase) as a model kinase and identified 23 novel candidate substrates for this kinase. Seven selected candidates were phosphorylated by p38 MAP kinase in vitro and in p38 MAP kinase‐activated cells. This proteomic approach can be applied to any protein kinase, allowing global identification of novel substrates.     

The Binding Mode of a Tau Peptide with Tubulin

The microtubule‐associated protein Tau promotes the polymerization of tubulin and modulates the function of microtubules. As a consequence of the dynamic nature of the Tau–tubulin interaction, the structural basis of this complex has remained largely elusive. By using NMR methods optimized for ligand–receptor interactions in combination with site‐directed mutagenesis we demonstrate that the flanking domain downstream of the four microtubule‐binding repeats of Tau binds competitively to a site on the α‐tubulin surface. The binding process is complex, involves partial coupling of different interacting regions, and is modulated by phosphorylation at Y394 and S396. This study strengthens the hypothesis of an intimate relationship between Tau phosphorylation and tubulin binding and highlights the power of the INPHARMA NMR method to characterize the interaction of peptides derived from intrinsically disordered proteins with their molecular partners.     

Optimized Tetrazine Derivatives for Rapid Bioorthogonal Decaging in Living Cells

The inverse‐electron‐demand Diels–Alder (iDA) reaction has recently been repurposed as a bioorthogonal decaging reaction by accelerating the elimination process after an initial cycloaddition between trans‐cyclooctene (TCO) and tetrazine (TZ). Herein, we systematically surveyed 3,6‐substituted TZ derivatives by using a fluorogenic TCO–coumarin reporter followed by LC‐MS analysis, which revealed that the initial iDA cycloaddition step was greatly accelerated by electron‐withdrawing groups (EWGs) while the subsequent elimination step was strongly suppressed by EWGs. In addition, smaller substituents facilitated the decaging process. These findings promoted us to design and test unsymmetric TZs bearing an EWG group and a small non‐EWG group at the 3‐ and 6‐position, respectively. These TZs showed remarkably enhanced decaging rates, enabling rapid iDA‐mediated protein activation in living cells.     

Mapping of phosphorylation sites in human MSK1 activated by a novel interaction with MRK‐β

The desire to map reliable phosphorylation signaling network has motivated the development of high‐performance techniques. Targeted biochemical studies and updated methods employing MS techniques are most used in mapping the phosphorylation sites and verifying novel interactions of kinases. Previously, we have established a novel method to efficiently facilitate more comprehensive, accurate phosphorylation site mapping of individual phosphoproteins by using combination of multiple stage MS analysis with target‐decoy database search against the much smaller targeted database. In this study, by applying this method, we have identified the phosphorylation sites in human MSK1 mitogen‐ and stress‐activated protein kinase 1), which has been proved to be a multi‐phosphorylated kinase that plays key roles in various cell functions, activated by a novel interaction with MRK‐β. The results show that this method can find out not only those previously identified active sites in MSK1, but also some novel phosphorylated sites, which correlates with biochemical evidence that, besides p38 and extracellular signal‐regulated kinase, MRK‐β could also activate MSK1 through direct interaction. Hence, we conclude this method is sensitive and reliable as expected and it can be further combined with automated screening and biochemical study in efficiently building up a more comprehensive phosphoprotein network.     

Engineered Substrate‐Specific Delta PKC Antagonists to Enhance Cardiac Therapeutics

Most protein kinases phosphorylate multiple substrates, each of which induces different and sometimes opposing functions. Determining the role of phosphorylation of each substrate following a specific stimulus is challenging but is essential to elucidate the role of that substrate in the signaling event. Here we describe a rational approach to identify inhibitors of delta protein kinase C (δPKC), each inhibiting the phosphorylation of only one of δPKC′s substrates. δPKC regulates many signaling events and we hypothesized that a docking inhibitor of a given substrate to δPKC should selectively abrogate the phosphorylation of only that substrate, without affecting the phosphorylation of the other δPKC substrates. Here we report the development of selective inhibitors of three δPKC substrates (in vitro K_d≈3 nm ); two greatly reduced ischemia‐induced cardiac injury with an IC₅₀ of ≈200 nm and the third had no effect, indicating that its respective substrate phosphorylation by δPKC has no role in the response to cardiac ischemia and reperfusion. The three inhibitors are highly specific; even at 1 μm , the phosphorylation of other δPKC protein substrates was unaffected. The rationale we describe is likely applicable for the development of other substrate‐specific inhibitors as well.     

Cross‐Linking/Mass Spectrometry for Studying Protein Structures and Protein–Protein Interactions: Where Are We Now and Where Should We Go from Here?

Structural mass spectrometry (MS) is gaining increasing importance for deriving valuable three‐dimensional structural information on proteins and protein complexes, and it complements existing techniques, such as NMR spectroscopy and X‐ray crystallography. Structural MS unites different MS‐based techniques, such as hydrogen/deuterium exchange, native MS, ion‐mobility MS, protein footprinting, and chemical cross‐linking/MS, and it allows fundamental questions in structural biology to be addressed. In this Minireview, I will focus on the cross‐linking/MS strategy. This method not only delivers tertiary structural information on proteins, but is also increasingly being used to decipher protein interaction networks, both in vitro and in vivo. Cross‐linking/MS is currently one of the most promising MS‐based approaches to derive structural information on very large and transient protein assemblies and intrinsically disordered proteins.     

High‐throughput workflow for identification of phosphorylated peptides by LC‐MALDI‐TOF/TOF‐MS coupled to in situ enrichment on MALDI plates functionalized by ion landing

We report an MS‐based workflow for identification of phosphorylated peptides from trypsinized protein mixtures and cell lysates that is suitable for high‐throughput sample analysis. The workflow is based on an in situ enrichment on matrix‐assisted laser desorption/ionization (MALDI) plates that were functionalized by TiO₂ using automated ion landing apparatus that can operate unsupervised. The MALDI plate can be functionalized by TiO₂ into any array of predefined geometry (here, 96 positions for samples and 24 for mass calibration standards) made compatible with a standard MALDI spotter and coupled with high‐performance liquid chromatography. The in situ MALDI plate enrichment was compared with a standard precolumn‐based separation and achieved comparable or better results than the standard method. The performance of this new workflow was demonstrated on a model mixture of proteins as well as on Jurkat cells lysates. The method showed improved signal‐to‐noise ratio in a single MS spectrum, which resulted in better identification by MS/MS and a subsequent database search. Using the workflow, we also found specific phosphorylations in Jurkat cells that were nonspecifically activated by phorbol 12‐myristate 13‐acetate. These phosphorylations concerned the mitogen‐activated protein kinase/extracellular signal‐regulated kinase signaling pathway and its targets and were in agreement with the current knowledge of this signaling cascade. Control sample of non‐activated cells was devoid of these phosphorylations. Overall, the presented analytical workflow is able to detect dynamic phosphorylation events in minimally processed mammalian cells while using only a short high‐performance liquid chromatography gradient. Copyright © 2015 John Wiley & Sons, Ltd.     

A Mix‐and‐Read Fluorescence Strategy for the Switch‐On Probing of Kinase Activity Based on an Aptameric‐Peptide/Graphene‐Oxide Platform

Protein kinase plays a vital role in regulating signal‐transduction pathways and its simple and quick detection is highly desirable because traditional kinase assays typically rely on a time‐consuming kinase‐phosphorylation process (ca. 1 h). Herein, we report a new and rapid fluorescence‐based sensing platform for probing the activity of protein kinase that is based on the super‐quenching capacity of graphene oxide (GO) nanosheets and specific recognition of the aptameric peptide (FITC‐IP₂₀). On the GO/peptide platform, the fluorescence quenching of FITC‐IP₂₀ that is adsorbed onto GO can be restored by selective binding of active protein kinase to the aptameric peptide, thereby resulting in the fast switch‐on detection of kinase activity (ca. 15 min). The feasibility of this method has been demonstrated by the sensitive measurement of the activity of cAMP‐dependent protein kinase (PKA), with a detection limit of 0.053 mU μL^?1. This assay technique was also successfully applied to the detection of kinase activation in cell lysate.     

Analysis of low‐density lipoprotein‐associated proteins using the method of digitized native protein mapping

The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel‐cutting, and quantitative LC‐MS/MS (in data‐independent acquisition mode, or MS^E), was improved by using a new MS/MS mode, ion mobility separation enhanced‐MS^E (HDMS^E), and applied to analyze the area of human plasma low‐density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid‐cut into 72 square gel pieces and subjected to quantitative LC‐MS/MS. Compared with MS^E, HDMS^E showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC‐HDMS^E and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B‐100 was the most abundant protein in the grid‐cut area, concentrated at pI ca. 5.4–6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39–42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B‐100. Twenty‐two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL.     

Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O‐PS of Vibrio cholerae O1, serotype Ogawa,and BSA revealed by matrix‐assisted laser desorption–ionization tandem mass spectrometry

We present the MALDI‐TOF/TOF‐MS analyses of various hapten–bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer‐equipped, terminal monosaccharide of the O‐specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten–BSA neoglycoconjugates with different hapten–BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI‐TOF/TOF‐MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI‐TOF/TOF‐MS/MS of the glycated peptides. The product‐ion scans of the protonated molecules were carried out with a MALDI‐TOF/TOF‐MS/MS tandem mass spectrometer equipped with a high‐collision energy cell. The high‐energy collision‐induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y‐series product ions was very useful for the sequencing of various peptides. The series of a‐ and b‐product ions confirmed the sequence of the conjugated peptides. Copyright © 2010 John Wiley & Sons, Ltd.     

A biuret‐derived,MS‐cleavable cross‐linking reagent for protein structural analysis: A proof‐of‐principle study

Chemical cross‐linking combined with mass spectrometry (XL‐MS) and computational modeling has evolved as an alternative method to derive protein 3D structures and to map protein interaction networks. Special focus has been laid recently on the development and application of cross‐linkers that are cleavable by collisional activation as they yield distinct signatures in tandem mass spectra. Building on our experiences with cross‐linkers containing an MS‐labile urea group, we now present the biuret‐based, CID‐MS/MS‐cleavable cross‐linker imidodicarbonyl diimidazole (IDDI) and demonstrate its applicability for protein cross‐linking studies based on the four model peptides angiotensin II, MRFA, substance P, and thymopentin.     

Thermal stabilization of tissues and the preservation of protein phosphorylation states for two‐dimensional gel electrophoresis

2‐DE is typically capable of discriminating proteins differing by a single phosphorylation or dephosphorylation event. However, a reliable representation of protein phosphorylation states as they occur in vivo requires that both phosphatases and kinases are rapidly and completely inactivated. Thermal stabilization of mouse cerebral cortex homogenates effectively inactivated these enzymes, as evidenced by comparison with unstabilized tissues where abscissal pI shifts were a common feature in 2‐D gels. Of the 588 matched proteins separated on 2‐D gels comparing stabilized and unstabilized tissues, 53 proteins exhibited greater than twofold differences in spot volume (ANOVA, p<0.05). Phosphoprotein‐specific staining was corroborated by the identification of 16 phosphoproteins by nano‐LC MS/MS and phosphotyrosine kinase activity assay.     

Global quantification of phosphoproteins combining metabolic labeling and gel‐based proteomics in B. pumilus

Differential proteomics targeting the protein abundance is commonly used to follow changes in biological systems. Differences in localization and degree of post‐translational modifications of proteins including phosphorylations are of tremendous interest due to the anticipated role in molecular regulatory processes. Because of their particular low abundance in prokaryotes, identification and quantification of protein phosphorylation is traditionally performed by either comparison of spot intensities on two‐dimensional gels after differential phosphoprotein staining or gel‐free by stable isotope labeling, sequential phosphopeptide enrichment and following LC‐MS analysis. In the current work, we combined in a proof‐of‐principle experiment these techniques using ¹⁴N/¹⁵N metabolic labeling with succeeding protein separation on 2D gels. The visualization of phosphorylations on protein level by differential staining was followed by protein identification and determination of phosphorylation sites and quantification by LC‐MS/MS. This approach should avoid disadvantages of traditional workflows, in particular the limited capability of peptide‐based gel‐free methods to quantify isoforms of proteins. Comparing control and stress conditions allowed for relative quantification in protein phosphorylation in Bacillus pumilus exposed to hydrogen peroxide. Altogether, we quantified with this method 19 putatively phosphorylated proteins.     

Identification of phosphoproteins and determination of phosphorylation sites by zirconium dioxide enrichment and SELDI-MS/MS

Reversible protein phosphorylation mediated by protein kinases and phosphatases is the most studied post-translational modification. Efficient characterization of phosphoproteomes is hampered by (1) low stoechiometry, (2) the dynamic nature of the phosphorylation process and (3) the difficulties of mass spectrometry to identify phosphoproteins from complex mixtures and to determine their sites of phosphorylation. Combination of the phosphopeptide enrichment method with MALDI-TOFMS, or alternatively, with HPLC-ESI-MS/MS and MS(3) analysis was shown to be a step forward for the successful application of MS in the study of protein phosphorylation. In our study we used phosphopeptide enrichment performed in a simple single-tube experiment using zirconium dioxide (ZrO(2)). A simple protein mixture containing precipitated bovine milk caseins was enzymatically digested and the mixture of tryptic fragments was analysed before and after enrichment using nanoflow HPLC-ESI-MS/MS and surface-enhanced laser desorption/ionization (SELDI)-MS/MS on QqTOF instruments to compare the efficiency of the two methods in the determination of phosphorylation sites. Both approaches confirm the high selectivity obtained by the use of batch-wise, ZrO(2)-based protocol using di-ammonium phosphate as the eluting buffer. More phosphorylation sites (five for beta-casein and three for alpha(S1)-casein) were characterized by SELDI-MS/MS than by nanoflow HPLC-ESI-MS/MS. Therefore, ZrO(2)-based phosphopeptide enrichment combined with SELDI-MS/MS is an attractive alternative to previously reported approaches for the study of protein phosphorylation in mixtures of low complexity with the advance of fast in situ peptide purification. The method was limited to successful analysis of high-abundance proteins. Only one phosphorylation site was determined for the minor casein component alpha(S2)-casein by ESI-MS/MS and none for kappa-casein. Therefore an improvement in enrichment efficiency, especially for successful phosphoproteomic applications, is needed.     

The Application of Fluorine‐Containing Reagents in Structural Proteomics

Structural proteomics refers to large‐scale mapping of protein structures in order to understand the relationship between protein sequence, structure, and function. Chemical labeling, in combination with mass‐spectrometry (MS) analysis, have emerged as powerful tools to enable a broad range of biological applications in structural proteomics. The key to success is a biocompatible reagent that modifies a protein without affecting its high‐order structure. Fluorine, well‐known to exert profound effects on the physical and chemical properties of reagents, should have an impact on structural proteomics. In this Minireview, we describe several fluorine‐containing reagents that can be applied in structural proteomics. We organize their applications around four MS‐based techniques: a) affinity labeling, b) activity‐based protein profiling (ABPP), c) protein footprinting, and d) protein cross‐linking. Our aim is to provide an overview of the research, development, and application of fluorine‐containing reagents in protein structural studies.     

The use of matrix coating assisted by an electric field (MCAEF) to enhance mass spectrometric imaging of human prostate cancer biomarkers

In this work, we combined a newly developed matrix coating technique – matrix coating assisted by an electric field (MCAEF) and matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) to enhance the imaging of peptides and proteins in tissue specimens of human prostate cancer. MCAEF increased the signal‐to‐noise ratios of the detected proteins by a factor of 2 to 5, and 232 signals were detected within the m/z 3500–37500 mass range on a time‐of‐flight mass spectrometer and with the sinapinic acid MALDI matrix. Among these species, three proteins (S100‐A9, S100‐A10, and S100‐A12) were only observed in the cancerous cell region and 14 proteins, including a fragment of mitogen‐activated protein kinase/extracellular signal‐regulated kinase kinase kinase 2, a fragment of cAMP‐regulated phosphoprotein 19, 3 apolipoproteins (C‐I, A‐I, and A‐II), 2 S100 proteins (A6 and A8), β‐microseminoprotein, tumor protein D52, α‐1‐acid glycoprotein 1, heat shock protein β‐1, prostate‐specific antigen, and 2 unidentified large peptides at m/z 5002.2 and 6704.2, showed significantly differential distributions at the p < 0.05 (t‐test) level between the cancerous and the noncancerous regions of the tissue. Among these 17 species, the distributions of apolipoprotein C‐I, S100‐A6, and S100‐A8 were verified by immunohistological staining. In summary, this study resulted in the imaging of the largest group of proteins in prostate cancer tissues by MALDI‐MS reported thus far, and is the first to show a correlation between S100 proteins and prostate cancer in a MS imaging study. The successful imaging of the three proteins only found in the cancerous tissues, as well as those showing differential expressions demonstrated the potential of MCAEF‐MALDI/MS for the in situ detection of potential cancer biomarkers. Copyright © 2015 John Wiley & Sons, Ltd.     

Probing kinase activities by electrochemistry, contact-angle measurements, and molecular-force interactions

Three different methods to investigate the activity of a protein kinase (casein kinase, CK2) are described. The phosphorylation of the sequence-specific peptide (1) by CK2 was monitored by electrochemical impedance spectroscopy (EIS). Phosphorylation of the peptide monolayer assembled on a Au electrode yields a negatively charged surface that electrostatically repels the negatively charged redox label [Fe(CN)6]3-/4-, thus increasing the interfacial electron-transfer resistance. The phosphorylation process by CK2 is further amplified by the association of the anti-phosphorylated peptide antibody to the monolayer. Binding of the antibody insulates the electrode surface, thus increasing the interfacial electron-transfer resistance in the presence of the redox label. This method enabled the quantitative analysis of the concentration of CK2 with a detection limit of ten units. The second method employed involved contact-angle measurements. Although the peptide 1-functionalized electrode revealed a contact angle of 67.5 degrees , phosphorylation of the peptide yielded a surface with enhanced hydrophilicity, 36.8 degrees. The biocatalyzed cleavage of the phosphate units with alkaline phosphatase regenerates the hydrophobic peptide monolayer, contact angle 55.3 degrees . The third method to characterize the CK2 system involved chemical force measurements between the phosphorylated peptide monolayer associated with the Au surface and a Au tip functionalized with the anti-phosphorylated peptide antibody. Although no significant rupture forces existed between the modified tip and the 1-functionalized surface (6+/-2 pN), significant rupture forces (multiples of 120+/-20 pN) were observed between the phosphorylated monolayer-modified surface and the antibody-functionalized tip. This rupture force is attributed to the dissociation of a simple binding event between the phosphorylated peptide and the fluorescent antibody (Fab) binding region.