Factors controlling the reactivity of zinc finger cores

Although the Zn(2+) cation in Zn·Cys(4), Zn·Cys(3)His, Zn·Cys(2)His(2), and Zn(2)Cys(6) cores of zinc finger (Zf) proteins typically plays a structural role, the Zn-bound thiolates in some Zf cores are reactive. Such labile Zf cores can serve as drug targets for retroviral or cancer therapies. Previous studies showed that the reactivity of a Zn-bound thiolate toward electrophiles is significantly reduced if it forms S---NH hydrogen bonds with the backbone amide. However, we found several well-known inactive Zf cores containing Cys ligands with no H-bonding interactions. Here, we show that H bonds from the peptide backbone or bonds from a second Zn cation to Zn-bound S atoms suppress the reactivity not only of these S atoms, but also of Zn-bound S* atoms with no interactions. Indeed, two or more indirect NH---S hydrogen bonds raise the free energy barrier for methylation of a Zn-bound S* in a Cys(4) core more than a direct NH---S* hydrogen bond. These findings help to elucidate why several well-known Zf cores have Cys ligands with no H bonds, but are unreactive. They also help to provide guidelines for distinguishing labile Cys-rich Zn sites from structural ones, which in turn help to identify novel potential Zf drug targets.     

利用高效液相色谱-串联质谱追踪酸奶发酵过程中的酪蛋白磷酸肽

酪蛋白磷酸肽(CPPs)是酪蛋白被磷酸化的片段,具有促进矿物元素吸收、抗氧化、预防龋齿等多种生物功能。该文利用高效液相色谱-串联质谱研究牛奶经嗜热链球菌(Streptococcus thermophilus)和保加利亚乳杆菌(Lactobacillus debrueckii subsp.bulgaricus)发酵后CPPs的释放规律。结果表明,在内源性蛋白酶的作用下牛奶含内源性CPPs,内源性CPPs主要来源于高丰度酪蛋白αs1-CN和β-CN。经乳酸菌发酵后,在乳酸菌蛋白酶的作用下更多酪蛋白的磷酸化位点暴露而产生大量CPPs,其中含SpSpSpEE特征结构的CPPs也在酸奶中检测到,CPPs的释放与酪蛋白结构密切相关。研究结果表明,牛奶经乳酸菌发酵后释放大量含特征结构SpSpSpEE的CPPs,可提高结合矿物元素的能力,从而增强其促进人体健康的生物功能。     

Sequencing of peptides phosphorylated on serines and threonines by post-source decay in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In the era of complete genome sequences, biochemical and medical research will focus more on the dynamic proteome of a cell. Regulation of proteins by post-translational modifications, which are not determined by the gene sequence, are already intensively studied. One example is phosphorylation of serines and threonines, probably the single most common cellular regulatory mechanism. In this paper we describe the sequencing of mono- and bisphosphorylated peptides, including identification of the phosphorylation sites, by post-source decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In addition to dephosphorylation of the parent ions, we studied the influence of the phosphate group on the fragmentation of peptides. Generally, peptides phosphorylated on serine and threonine residues displayed no difference in their fragmentation patterns. The intensities of the resulting fragment ion signals depend only on the peptide sequence and not on either the phosphorylated amino acid or its position in the peptide chain. Phosphorylation increased the bond cleavage C-terminal to the phosphorylation site more than 10-fold, resulting in abundant signals, which typically dominated the PSD spectra. The produced C-terminally phosphorylated b-type fragment ions showed characteristic dephosphorylated fragment ions b(n) -H(3)PO(4) (-98 Da) and b(n) -HPO(3) (-80 Da) of higher abundances than the phosphorylated fragment ion. As a second layer to identify the phosphorylation site, all internally phosphorylated fragment ions were accompanied by minor, but always detectable, signals of the dephosphorylated fragment ions. Interpretation of PSD spectra of phosphopeptides was not more complicated than for unphosphorylated peptides, despite the increased number of obtained fragment ion signals.     

Dynamic identification of phosphopeptides using immobilized metal ion affinity chromatography enrichment, subsequent partial beta-elimination/chemical tagging and matrix-assisted laser desorption/ionization mass spectrometric analysis

The enrichment of phosphopeptides using immobilized metal ion affinity chromatography (IMAC) and subsequent mass spectrometric analysis is a powerful protocol for detecting phosphopeptides and analyzing their phosphorylation state. However, nonspecific binding peptides, such as acidic, nonphosphorylated peptides, often coelute and make analyses of mass spectra difficult. This study used a partial chemical tagging reaction of a phosphopeptide mixture, enriched by IMAC and contaminated with nonspecific binding peptides, following a modified beta-elimination/Michael addition method, and dynamic mass analysis of the resulting peptide pool. Mercaptoethanol was used as a chemical tag and nitrilotriacetic acid (NTA) immobilized on Sepharose beads was used for IMAC enrichment. The time-dependent dynamic mass analysis of the partially tagged reaction mixture detected intact phosphopeptides and their mercaptoethanol-tagged derivatives simultaneously by their mass difference (-20 Da for each phosphorylation site). The number of new peaks appearing with the mass shift gave the number of multiply phosphorylated sites in a phosphopeptide. Therefore, this partial chemical tagging/dynamic mass analysis method can be a powerful tool for rapid and efficient phosphopeptide identification and analysis of the phosphorylation state concurrently using only MS analysis data.     

Effect of Zn...Zn separation on the hydrolytic activity of model dizinc phosphodiesterases

From the study of highly preorganized model systems, experimental support has been obtained for a possible functional role of the Zn-(H)O...HO(H)-Zn motif in oligozinc hydrolases. The mechanistic relevance of such an array, which may be described as a hydrated form of a pseudo-terminal Zn-bound hydroxide, has recently been supported by DFT calculations on various metallohydrolase active sites. In the present targeted approach, the Zn...Zn distance in two related dizinc complexes has been controlled through the use of multifunctional pyrazolate-based ligand scaffolds, giving either a tightly bridged Zn-O(H)-Zn or a more loosely bridged Zn-(H)O...HO(H)-Zn species in the solid state. Zn-bound water has been found to exhibit comparable acidity irrespective of whether the resulting hydroxide is supported by strong hydrogen-bonding in the O(2)H(3) moiety or is in a bridging position between two zinc ions, indicating that water does not necessarily have to adopt a bridging position in order for its pK(a) to be sufficiently lowered so as to provide a Zn-bound hydroxide at physiological pH. Comparative reactivity studies on the cleavage of bis(4-nitrophenyl)phosphate (BNPP) mediated by the two dizinc complexes have revealed that the system with the larger Zn...Zn separation is hydrolytically more potent, both in the hydrolysis and the transesterification of BNPP. The extent of active site inhibition by the reaction products has also been found to be governed by the Zn...Zn distance, since phosphate diester coordination in a bridging mode within the clamp of two zinc ions is only favored for Zn...Zn distances well above 4 A. Different binding affinities are rationalized in terms of the structural characteristics of the product-inhibited complexes for the two different ligand scaffolds, with dimethyl phosphate found as a bridging ligand within the bimetallic pocket.     

Phosphopeptide elution times in reversed-phase liquid chromatography

Elution time shifts between 33 different peptides and their corresponding phosphopeptides ranging from 4 amino acid residues to 35 amino acids in length were systematically investigated using high-resolution reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) analysis with trifluoroacetic acid as the ion pairing agent. Observed peptide elution time shifts for a single phosphorylation ranged from -5.28 min (for pYVPML) to +0.59 min (for HRDpSGLLDSLGR). Peptides containing a phosphotyrosine residue displayed a significant decrease in elution time following phosphorylation compared to their similar-sized peptides with phosphoserine or phosphothreonine residues. While peptide phosphorylation generally led to a decrease in the observed elution time, five peptides displayed increased elution times as a result of phosphorylation. For large peptides (> or =18 amino acids), the elution time shifts due to single phosphorylation were limited (ranging between -0.48 and +0.03 min), while the elution time shifts for small peptides (<18 amino acids) were characterized by a larger deviation (ranging between -5.28 and +0.59 min). The predictive capability for the observed RPLC elution time change due to phosphorylation has been suggested, which will aid in assigning confident phosphopeptide identifications and their subsequent confirmation.     

蛋白质组衍生肽段文库用于鉴定酪蛋白激酶2的底物模体

模体是蛋白质二级结构上的一种特征序列,激酶底物通常具有一类特征性的模体,识别底物模体对鉴定激酶底物具有重要意义。为了快速鉴定激酶底物模体,将全细胞蛋白质酶解液作为肽段文库的来源,采用碱性磷酸酶去除肽段的固有磷酸化后构建了用于筛选激酶底物模体的肽段文库。该肽段文库是大量非磷酸化肽段的混合物,将此混合肽段与酪蛋白激酶2和三磷酸腺苷作用30 min后,通过固定化金属离子亲和色谱法富集磷酸化肽段,采用反相液相色谱-串联质谱分析,成功地鉴定到472条非冗余底物肽段,包含451个非冗余磷酸化位点,并由此得到底物模体S/T-D/E-x-D/E。该法能够快速准确地筛选出激酶底物模体,对研究激酶-底物识别以及信号转导过程具有重要意义。     

A binary matrix for improved detection of phosphopeptides in matrix‐assisted laser desorption/ionization mass spectrometry

Application of matrix‐assisted laser‐desorption/ionization mass spectrometry (MALDI MS) to analysis and characterization of phosphopeptides in peptide mixtures may have a limitation, because of the lower ionizing efficiency of phosphopeptides than nonphosphorylated peptides in MALDI MS. In this work, a binary matrix that consists of two conventional matrices of 3‐hydroxypicolinic acid (3‐HPA) and α‐cyano‐4‐hydroxycinnamic acid (CCA) was tested for phosphopeptide analysis. 3‐HPA and CCA were found to be hot matrices, and 3‐HPA not as good as CCA and 2,5‐dihydroxybenzoic acid (DHB) for peptide analysis. However, the presence of 3‐HPA in the CCA solution with a volume ratio of 1:1 could significantly enhance ion signals for phosphopeptides in both positive‐ion and negative‐ion detection modes compared with the use of pure CCA or DHB, the most common phosphopeptide matrices. Higher signal intensities of phosphopeptides could be obtained with lower laser power using the binary matrix. Neutral loss of the phosphate group (?80 Da) and phosphoric acid (?98 Da) from the phosphorylated‐residue‐containing peptide ions with the binary matrix was decreased compared with CCA alone. In addition, since the crystal shape prepared with the binary matrix was more homogeneous than that prepared with DHB, searching for ‘sweet’ spots can be avoided. The sensitivity to detect singly or doubly phosphorylated peptides in peptide mixtures was higher than that obtained with pure CCA and as good as that obtained using DHB. We also used the binary matrix to detect the in‐solution tryptic digest of the crude casein extracted from commercially available low fat milk sample, and found six phosphopeptides to match the digestion products of casein, based on mass‐to‐charge values and LIFT TOF‐TOF spectra. Copyright © 2009 John Wiley & Sons, Ltd.     

Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis

Large-scale characterization of phosphoproteins requires highly specific methods for the purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed. Femtomole phosphopeptides from digestion products could be enriched by ZrO2 nanoparticles and can be well detected by MALDI mass spectrometric analysis. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano-LC-MS/MS) and MS/MS/MS. A total of 248 defining phosphorylation sites and 140 phosphorylated peptides were identified by manual validation using a series of rigid criteria.     

Differential effects of the Zn-His-Bkb vs Zn-His-[Asp/Glu] triad on Zn-core stability and reactivity

The most common partner of the Zn-bound His is the Asp/Glu carboxylate side chain in catalytic Zn sites and the backbone (Bkb) carbonyl group in structural Zn sites. To elucidate the factors governing the selection of the second-shell partner of the Zn-bound His in structural/catalytic Zn sites, systematic studies using density functional theory and continuum dielectric calculations were performed to determine the relative contributions of the second-shell Bkb carbonyl and the Asp/Glu carboxylate to the Zn-core stability and reactivity. The results show that the contributions of the second-shell Bkb carbonyl and Asp/Glu carboxylate to the Zn-core stability depend mainly on the solvent accessibility of the Zn-site and the composition of the Zn-core. They reveal the advantage of a second-shell Bkb carbonyl in anionic Zn cavities: it stabilizes anionic, buried Zn-cores more than the corresponding negatively charged Asp/Glu carboxylate, thus explaining the absence of the Zn-His-Asp/Glu triad in structural [Zn(Cys)3(His)]- cores. They also reveal the advantage of a second-shell Asp/Glu carboxylate in catalytic Zn-cores: relative to a Bkb carbonyl group, it increases (i) the HOMO energy of the cationic/neutral zinc core, (ii) the reactivity of the attacking Zn-bound OH-, (iii) electron transfer to the substrate, and (iv) the stability of the metal complex upon electron transfer. Furthermore, a second-shell Asp/Glu carboxylate could facilitate product release in the common cationic catalytic cores, by acting as a proton acceptor of the Zn-bound His creating an Asp...His- dyad that stabilizes the zinc dication more than the respective Bkb...His0 dyad.     

The effect of phosphorylation on the electron capture dissociation of peptide ions

The effect of site and frequency of phosphorylation on the electron capture dissociation of peptide ions has been investigated. The ECD of a suite of synthetic peptides (APLSFRGSLPKSYVK; one unmodified, three singly-phosphorylated, three-doubly phosphorylated, and one triply-phosphorylated); two tryptic phosphopeptides (YKVPQLEIVPN(p)SAEER, alpha-casein and FQ(p)SEEQQQTEDELQDK, beta-casein) and their unmodified counterparts, were determined over a range of ECD cathode potentials. The results show that, for doubly-charged precursor ions, the presence of phosphorylation has a deleterious effect on ECD sequence coverage. The fragmentation patterns observed suggest that for peptides with multiple basic residues, the phospho-groups exist in their deprotonated form and form salt-bridges with protonated amino acid side chains. The fragmentation observed for the acidic tryptic peptides suggested the presence of noncovalent interactions, which were perturbed on phosphorylation. Increasing the ECD electron energy significantly improves sequence coverage. Alternatively, improved sequence coverage can be achieved by performing ECD on triply-charged precursor ions. The findings are important for the understanding of gas-phase fragmentation of phosphopeptides.     

Molecular recognition and fluorescence sensing of monophosphorylated peptides in aqueous solution by bis(zinc(II)-dipicolylamine)-based artificial receptors

The phosphorylation of proteins represents a ubiquitous mechanism for the cellular signal control of many different processes, and thus selective recognition and sensing of phosphorylated peptides and proteins in aqueous solution should be regarded as important targets in the research field of molecular recognition. We now describe the design of fluorescent chemosensors bearing two zinc ions coordinated to distinct dipicolylamine (Dpa) sites. Fluorescence titration experiments show the selective and strong binding toward phosphate derivatives in aqueous solution. On the basis of (1)H NMR and (31)P NMR studies, and the single-crystal X-ray structural analysis, it is clear that two Zn(Dpa) units of the binuclear receptors cooperatively act to bind a phosphate site of these derivatives. Good agreement of the binding affinity estimated by isothermal titration calorimetry with fluorescence titration measurements revealed that these two receptors can fluorometrically sense several phosphorylated peptides that have consensus sequences modified with natural kinases. These chemosensors display the following significant features: (i) clear distinction between phosphorylated and nonphosphorylated peptides, (ii) sequence-dependent recognition, and (iii) strong binding to a negatively charged phosphorylated peptide, all of which can be mainly ascribed to coordination chemistry and electrostatic interactions between the receptors and the corresponding peptides. Detailed titration experiments clarified that the phosphate anion-assisted coordination of the second Zn(II) to the binuclear receptors is crucial for the fluorescence intensification upon binding to the phosphorylated derivatives. In addition, it is demonstrated that the binuclear receptors can be useful for the convenient fluorescent detection of a natural phosphatase (PTP1B) catalyzed dephosphorylation.     

Evaluation of the titanium dioxide approach for MS analysis of phosphopeptides

The affinity of titanium dioxide for phosphate groups has been successfully used for enrichment of phosphopeptides from complex mixtures. This paper reports the relationship between the occurrence of some amino acids and the phospho-specific and nonspecific binding of peptides that occurs during titanium dioxide enrichment. In order to perform a systematic study, two well-characterized peptide mixtures consisting of either 33 or 8 synthetic phosphopeptides and their nonphosphorylated analogs, which differed in charge and hydrophobicity, were synthesized and analyzed by ESI-MS and MALDI-MS. The titanium dioxide procedure was also evaluated for comprehensive detection of phosphopeptides in phosphoproteomics. In summary, our results clearly confirm the high selectivity of titanium dioxide for phosphorylated sequences. Drastically reduced recovery was observed for phosphopeptides with multiple basic amino acids. Nonspecific binding of nonphosphorylated peptides and sample loss of phosphopeptides must also be taken into account.     

C60-fullerene bound silica for the preconcentration and the fractionation of multiphosphorylated peptides

Phosphorylation of proteins is an important cellular regulatory process. The analysis of protein phosphorylation is challenging due to the high dynamic range and low abundance natures of phosphorylated species. Mass spectrometry (MS) of phosphopeptides obtained from tryptic protein digests is the method-of-choice for characterization of phosphorylated proteins. However, determination of phosphopeptides by MS represents a major challenge, especially in the presence of unmodified peptides. Due to lower ionization efficiency of phosphopeptides, as well as the fact that the stoichiometry of phosphorylation is often present at low relative abundance, efficient enrichment of the phosphorylated peptides prior to MS analysis is therefore of high demand. In addition, successful identification of peptides with different phosphorylation grades still remains challenging.     

Inverted micelle formation of cell-penetrating peptide studied by coarse-grained simulation: importance of attractive force between cell-penetrating peptides and lipid head group

Arginine-rich peptide and Antennapedia are cell-penetrating peptides (CPPs) which have the ability to permeate plasma membrane. Deformation of the plasma membrane with CPPs is the key to understand permeation mechanism. We investigate the dynamics of CPP and the lipid bilayer membrane by coarse-grained simulation. We found that the peptide makes inverted micelle in the lipid bilayer membrane, when the attractive potential between the peptide and lipid heads is strong. The inverted micelle is formed to minimize potential energy of the peptide. For vesicle membrane, the peptide moves from the outer vesicle to the inner vesicle through the membrane. The translocation of the peptide suggests inverted micelle model as a possible mechanism of CPPs.     

纳升级电喷雾离子源-四极杆-飞行时间质谱中磷酸化肽的离子抑制作用

目前磷酸化蛋白质组学研究中的主要技术是蛋白质酶解产生的磷酸化肽的质谱检测。但是实际样品中的磷酸化肽(特别是多磷酸化肽)很难被检测到。其原因普遍认为是由于质谱检测时,非磷酸化肽抑制磷酸化肽。但也有认为非磷酸化肽对磷酸化肽没有抑制作用。另外磷酸化肽之间是否存在离子抑制作用还没有报道。本文采用相同氨基酸序列的标准磷酸化肽和非磷酸化肽,将其单独和混合进行质谱检测,通过对比混合前后磷酸化肽的信号强度,证明了非磷酸化肽对磷酸化肽有离子抑制作用;单磷酸化肽对二磷酸化肽有一定的抑制作用,但不太显著;单磷酸化肽对三磷酸化肽、二磷酸化肽对三磷酸化肽均有显著的离子抑制作用。该研究为今后单磷酸化肽和多磷酸化肽的分段富集和检测提供了有力的证明。     

Identification of phosphorylation sites of equine β‐casein isoforms

Equine β‐casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P–7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion‐exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium dioxide immobilized onto a micro‐column. This method turned out to be an efficient tool to separate the CPPs Arg¹–Lys³⁴ and Glu⁴–Lys³⁴ from non‐phosphorylated peptides. Purification was achieved by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and each CPP was hydrolyzed by endoproteinase Glu‐C. Finally, the digests were analyzed by RP‐HPLC/electrospray ionization mass spectrometry (RP‐HPLC/ESI‐MS) and identified by nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) to locate the phosphorylated sites of the β‐casein isoforms 4P–7P with accuracy. Thus, the isoform 4P was found to be phosphorylated on residues Ser⁹, Ser²³, Ser²⁴, and Ser²⁵. Addition of phosphate groups on Ser¹⁸, Thr¹², and Ser¹⁰ led to the formation of the isoforms 5P–7P, respectively. The results indicated that the in vivo phosphorylation of the equine β‐casein follows a sequential way and is not randomly performed. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns for selective enrichment of phosphopeptides

In this study, a novel method for the highly selective enrichment of phosphopeptides using erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns is presented. Erbium phosphate was synthesized by precipitation from boiling phosphoric acid and incubated overnight in erbium chloride solutions. The resulting powder was embedded in a monolithic poly(glycidyl methacrylate/ethylene dimethacrylate) polymer. The monolith was synthesized in a spin column by radical polymerization. Erbium phosphate demonstrated a high affinity and selectivity for phosphopeptides due to the strong interaction of trivalent erbium ions with the phosphate groups of phosphopeptides. The high selectivity and performance of the designed spin columns were demonstrated by successfully enriching phosphopeptides from tryptically digested protein mixtures containing the model phosphoproteins α‐ and β‐casein, bovine milk, and human saliva. By the implementation of several washing steps, unspecific components were removed and the enriched phosphopeptides were effectively eluted from the spin columns under alkaline conditions. The selective performance of the presented method was further demonstrated by the enrichment of two synthetic phosphopeptides, which were spiked in tryptically digested and dephosphorylated HeLa cell lysates at low ratios. Finally, the presented approach was compared to conventional phosphopeptide enrichment by titanium oxide and revealed higher recoveries for the erbium phosphate doped monoliths.     

Cyclic phosphopeptides for interference with Grb2 SH2 domain signal transduction prepared by ring-closing metathesis and phosphorylation

Cyclic phosphopeptides were prepared using ring-closing metathesis followed by phosphorylation. These cyclic phosphopeptides were designed to interact with the SH2 domain of Grb2, which is a signal transduction protein of importance as a target for antiproliferative drug development. Binding of these peptides to the Grb2 SH2 domain was evaluated by a surface plasmon resonance assay. High affinity binding to the Grb2 SH2 domain was maintained upon macrocyclization, thus indicating that this method can be used to assemble high affinity cyclic phosphopeptides that interfere with signal transduction cascades.     

Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used successfully to detect phosphorylation sites in proteins. Applications may be limited by the low response of phosphopeptides compared to nonphosphorylated peptides in MALDI MS. The addition of ammonium salts to the matrix/analyte solution substantially enhances the signal for phosphopeptides. In examples shown for equimolar mixtures, the phosphorylated peptide peaks become the largest peaks in the spectrum upon ammonium ion addition. This can allow for the identification of phosphopeptides in an unfractionated proteolytic digestion mixture. Sufficient numbers of protonated phosphopeptides can be generated such that they can be subjected to postsource decay analysis, in order to confirm the number of phosphate groups present. The approach works well with the common MALDI matrices such as α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid, and with ammonium salts such as diammonium citrate and ammonium acetate.