Sequence-dependent enrichment of a model phosphopeptide: a combined MALDI-TOF and NMR study

The goal of this study was to detect and quantify by MALDI-TOF MS the phosphorylation of a peptide containing the recognition motif of the Protein Kinase C (PKC). Such model peptide can be used as a phosphorylation probe to follow intracellular kinase/phosphatase activities. This study allowed us to establish relationships between sequence specificities and affinity for TiO(2) or IMAC media. The peptide has the sequence biotin-GGGGCFRTPSFLKK-NH(2) in which the serine residue can be phosphorylated. Enrichment of the corresponding phosphopeptide, by the dedicated IMAC and TiO(2) affinity chromatography methods, proved inefficient. By combining MALDI-TOF and NMR data, we first showed that the lack of affinity of the phosphopeptide for TiO(2) was partly related to the basic property of its peptide sequence. Furthermore, the peptide shows local structuration around the P(9)- S(10) segment, with formation of a salt bridge between the guanidinium group of the R(7) side chain and the phosphate moiety. In conjunction with an inadequate position of the {biotin-G(4)} N-terminal tag, this local structure could shield the phosphate group, preventing interaction with TiO(2). To improve TiO(2) affinity, the peptide sequence was modified accordingly. The new sequences retained the biological properties while their enrichment by IMAC or TiO(2) became possible.     

Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies

Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A–class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg^2.39, His^2.43 and Glu^3.46, which makes a polar lock with T^6.37. These alignments and models provide useful tools for understanding class B GPCR function.     

Combining mass spectrometry and pull-down techniques for the study of receptor heteromerization. Direct epitope-epitope electrostatic interactions between adenosine A2A and dopamine D2 receptors

Previous results from FRET and BRET experiments and computational analysis (docking simulations) have suggested that a portion of the third intracellular loop (I3) of the human dopamine D2 receptor (D2R) and the C-tail from the human adenosine A2A receptor (A2AR) are involved in A2AR-D2R heteromerization. The results of the present studies, using pull-down and mass spectrometry experiments, suggest that A2AR-D2R heteromerization depends on an electrostatic interaction between an Arg-rich epitope from the I3 of the D2R (217RRRRKR222) and two adjacent Asp residues (DD401-402) or a phosphorylated Ser (S374) residue in the C-tail of the A2AR. A GST-fusion protein containing the C-terminal domain of the A2AR (GST-A2ACT) was able to pull down the whole D2R solubilized from D2R-tranfected HEK-293 cells. Second, a peptide corresponding to the Arg-rich I3 region of the D2R (215VLRRRRKRVN224) and bound to Sepharose was able to pull down both GST-A2ACT and the whole A2AR solubilized from A2AR-tranfected HEK-293 cells. Finally, mass spectometry and pull-down data showed that the Arg-rich D2R epitope binds to two different epitopes from the C-terminal part of the A2AR, containing the two adjacent Asp residues or the phosphorylated Ser residue (388HELKGVCPEPPGLDDPLAQDGAVGS412 and 370SAQEpSQGNT378). The present results are the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization, a new, expanding area of protein-protein interactions.     

Single color fluorescent indicators of protein phosphorylation for multicolor imaging of intracellular signal flow dynamics

Existing monitoring methods for protein phosphorylation involved in intracellular signal transduction in vivo are exclusively based on fluorescence resonance energy transfer, which needs the measurement of the change in fluorescence intensities at two wavelengths. Therefore, it is difficult to monitor protein phosphorylation together with other related signaling processes, such as second messengers and protein translocation. To overcome this problem, we developed novel fluorescent indicators, each containing a differently colored (cyan and green) single fluorophore. The present indicator is a tandem fusion protein containing a kinase substrate domain, a circularly permuted fluorescent protein (cpFP), and a phosphorylation recognition domain. The cpFP is obtained by dividing a green fluorescent protein mutant (GFP) at residue 144-145 and linking the carboxy and amino portions thereof with a peptide linker. The substrate domain used in this study is a peptide sequence that is phosphorylated by insulin receptor. Phosphorylation of the substrate domain induces its interaction with the phosphorylation recognition domain, which causes a conformational change in the cpFP and a change in its fluorescence. The cyan and green indicators exhibited 10% decrease and 15% increase, respectively, in their fluorescence intensities upon phosphorylation. Using this cyan indicator and GFP-tagged mitogen-activated protein kinase (MAPK), we found that insulin-induced protein phosphorylation occurred immediately upon the addition of insulin, whereas nuclear translocation of MAPK occurred 7 min later. By tailoring the substrate domains and the phosphorylation recognition domains in these cyan and green indicators, the present approach should be applicable to the in vivo analysis of a broad range of protein phosphorylation processes, together with other intracellular signaling processes.     

Hydrophobic interaction chromatography of soluble interleukin I receptor type II to reveal chemical degradations resulting in loss of potency

A hydrophobic interaction chromatography method was developed to analyze recombinant soluble Interleukin 1 receptor type II (sIL-1R type II) drug substance and assess the stability of the drug under accelerated degradation studies. HIC resolved the degraded molecules into three peaks. A combination of several analytical techniques, including cyanogen bromide cleavage, reversed-phase chromatography, mass spectrometry, and N-terminal sequencing, were used to identify the origins of these peaks. We found that accelerated degradation resulted from three different events, deamidation and isomerization at asparagine 317 (Asn317), C-terminal cleavage, and aggregation. The iso-aspartate 317 (iso-Asp317)-containing species were shown to elute in HIC peak I and the Asp317-containing species in HIC peak II, respectively. Deamidation-isomerization to iso-Asp317, but not deamidation to Asp317, resulted in altered retention time on HIC companied by loss of potency, presumably by introducing a significant conformational change. CNBr C-terminal analysis showed that the inactive HIC peak I consisted of sIL-1R type II with "large" C-terminal truncations of 13 or 14 amino acids, whereas the active HIC peak II contained C-terminally full length and "small" C-terminal clips of two amino acids. Molecular modeling indicates that the short loop D317-S320, in the third domain of IL-1R type II, has a crucial impact on the stability of the molecule.     

Identification of phosphorylation sites in insulin receptor substrate-1 by hypothesis-driven high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) can regulate tyrosine phosphorylation of IRS-1 and subsequent insulin signaling. The 182 serine and 60 threonine residues in IRS-1 make position-by-position analysis of potential phosphorylation sites by mutagenesis difficult. Tandem mass spectrometry provides a more efficient way to identify phosphorylated residues in IRS-1. Toward this end, we overexpressed glutathione S-transferase-IRS-1 fusion proteins in E. coli and treated them in vitro with various kinases followed by identification of phosphorylation sites using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Nine phosphorylation sites were detected in the tryptic digests of middle and C-terminal regions of IRS-1 treated with protein kinase A or extracellular signal-regulated kinase 2. Of these sites, five have not previously been detected by any method and provide novel candidates for identification in cells or in vivo.     

Computational modelling reveals feedback redundancy within the epidermal growth factor receptor/extracellular-signal regulated kinase signalling pathway

The epidermal growth factor receptor (EGFR) activated extracellular-signal regulated kinase (ERK) pathway is a central cell signalling pathway that mediates many biological responses including cell proliferation, transformation, survival and motility. Deregulation of the pathway either through mutation of components or overexpression of EGFRs is associated with several forms of cancer. Under normal conditions, EGF stimulates a rapid but transient activation of ERK as the signal is rapidly shutdown, whereas under cancerous conditions, the ERK signal cannot be shutdown and is sustained. Computational modelling techniques have been used to investigate the signalling dynamics of the EGFR/ERK pathway, focusing on identifying the key processes involved in signal termination and what role the ERK to son of sevenless (SOS) negative feedback loop plays in generating a transient response. This model predicts that this negative feedback loop is not needed to achieve a transient activation of ERK as the process of receptor degradation alone is enough to terminate the signal. Importantly, the behaviour and predictions of this model are verified with laboratory data, as is essential for modern systems biology approaches. Further analysis showed that the feedback loop and receptor degradation were both redundant processes, as each could compensate for the absence of the other. This led to the prediction that in the case of a receptor which is not degraded, such as the insulin receptor, the negative feedback loop to SOS will actually be essential for a transient response to be achieved. Overall, the results shed new light on the role of negative feedback in EGF receptor signalling and suggest that different receptors are dependent on different features within the ERK pathway when relaying their signals.     

G-protein-coupled receptor chromatographic stationary phases. 2. Ligand-induced conformational mobility in an immobilized beta2-adrenergic receptor

Membranes from a HEK-293 cell line expressing the beta(2)-adrenergic receptor (beta(2)-AR) have been immobilized on an artificial membrane liquid chromatographic stationary phase. The resulting phase was packed into a glass column (1.8 x 0.5 (i.d.) cm) and used in on-line chromatographic system. Frontal displacement affinity chromatography was used to determine the dissociation constants (K(d)) of CGP 12177A (552.6 nM) and (S)-propranolol (84.3 nM). Zonal displacement chromatography using CGP 12177A as the marker and racemic mixtures of the antagonists nadolol and propranolol demonstrated that the immobilized beta(2)-AR retained its ability to specifically bind these compounds. Similar experiments with (R)- and (S)-propranolol demonstrated that the immobilized receptor retained its enantioselectivity as (S)-propranolol displaced the CGP 12177 marker to a great extent that the (R)-enantiomer. The addition of the agonist butoxamine to the mobile phase increased the retention of the CGP-12177A as did the addition of the agonist fenoterol. These results indicate that the immobilized beta(2)-AR retained its ability to undergo ligand-induced conformational changes. The data from this study suggest that the immobilized beta(2)-AR can be used to screen for ligand binding interactions in both the resting and active states of the receptor.     

CSF－1R部分基因在大肠杆菌中表达、抗融合蛋白血清制备及丝氨酸／苏氨酸磷酸化的初步研究

鼠巨噬细胞集落刺激因子－１受体（ｍＣＳＦ－１Ｒ）部分序列与质粒ｐＧＥＸ－２Ｔ谷胱苷肽转移酶（ＧＳＴ）融合，融合蛋白ＧＳＴ－ＣＤ－Ｐｓｔ（胞浆区），ＧＳＴ－ＣＴｅｒｍ（Ｃ－末端）和ＧＳＴ－ＫＩ（激酶插入区）成功地在大肠杆菌ＪＭ１０９株表达。初步结果指出：（１）ＧＳＴ－融合蛋白在体外激酶分析中可以作为底物；（２）由ＰＫＡ导致的磷酸化可能具有生理学意义；（３）ｍＣＳＦ－１Ｒ被ＣＫＩＩ磷酸化。３２Ｐ标记ＧＳＴ－ＣＤ－Ｐｓｔ的磷酸氨基酸分析证实，ｍＣＳＦ－１Ｒ的胞浆区丝氨酸上被磷酸化，已制备抗ＧＳＴ－ＣＴｅｒｍ，ＧＳＴ－ＫＩ和ＧＳＴ－ＣＤ－Ｐｓｔ兔抗体。抗血清的筛选通过野生型３２Ｄ－ＣＳＦ－１Ｒ转染子免疫沉淀进行。     

Biarsenical-tetracysteine motif as a fluorescent tag for detection in capillary electrophoresis

Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, relatively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine whether the biarsenical labeling scheme could be extended to fluorescent detection of analytes in capillary electrophoresis. Recombinant protein or synthesized peptides containing the optimized tetracysteine motif "-C-C-P-G-C-C-" were labeled with biarsenical dyes and then analyzed by micellar electrokinetic capillary chromatography (MEKC). The biarsenical-tetracysteine complex was stable and remained fluorescent under standard MEKC conditions for peptide and protein separations. The detection limit following electrophoresis in a capillary was less than 3 x 10(-20) mol with a simple laser-induced fluorescence system. A mixture of multiple biarsenical-labeled peptides and a protein were easily resolved. Demonstrating that the label did not interfere with bioactivity, a peptide-based enzyme substrate conjugated to the tetracysteine motif and labeled with a biarsenical dye retained its ability to be phosphorylated by the parent kinase. The feasibility of using this label for chemical cytometry experiments was shown by intracellular labeling and subsequent analysis of a recombinant protein possessing the tetracysteine motif expressed in living cells. The extension of the biarsenical-tetracysteine tag to fluorescent labeling of peptides and proteins in chemical separations is a valuable addition to biochemical and cell-based investigations.     

胰岛素受体酪氨酸蛋白激酶对醛糖还原酶的磷酸化作用

从人胎盘纯化了醛糖还原酶和部分纯化了胰岛素受体。通过磷酸化实验证明：醛糖还原酶是胰岛素受体酪氨酸蛋白激酶的底物，经分析揭示：胰岛素受体酪氨酸蛋白激酶可将醛糖还原酶的第４０位和第４９位的酪氨酸残基磷酸化。在试管内，未磷酸化的醛糖还原酶和磷酸化的醛糖还原酶的动力学常数无差异，而细胞内的情况有待于进一步研究，本实验提示：醛糖还原酶除具酶活性外，可能还有在细胞内传递胰腺素信息的功能。     

On the organization of human T-cell receptor loci: log-periodic distribution of T-cell receptor gene segments

The human T-cell repertoire is complex and is generated by the rearrangement of variable (V), diversity (D) and joining (J) segments on the T-cell receptor (TCR) loci. The T-cell repertoire demonstrates self-similarity in terms clonal frequencies when defined by V, D and J gene segment usage; therefore to determine whether the structural ordering of these gene segments on the TCR loci contributes to the observed clonal frequencies, the TCR loci were examined for self-similarity and periodicity in terms of gene segment organization. Logarithmic transformation of numeric sequence order demonstrated that the V and J gene segments for both T-cell receptor α (TRA) and β (TRB) loci are arranged in a self-similar manner when the spacing between the adjacent segments was considered as a function of the size of the neighbouring gene segment, with an average fractal dimension of approximately 1.5. Accounting for the gene segments occurring on helical DNA molecules with a logarithmic distribution, sine and cosine functions of the log-transformed angular coordinates of the start and stop nucleotides of successive TCR gene segments showed an ordered progression from the 5′ to the 3′ end of the locus, supporting a log-periodic organization. T-cell clonal frequency estimates, based on V and J segment usage, from normal stem cell donors were plotted against the V and J segment on TRB locus and demonstrated a periodic distribution. We hypothesize that this quasi-periodic variation in gene-segment representation in the T-cell clonal repertoire may be influenced by the location of the gene segments on the periodic-logarithmically scaled TCR loci. Interactions between the two strands of DNA in the double helix may influence the probability of gene segment usage by means of either constructive or destructive interference resulting from the superposition of the two helices.     

Analysis of protein phosphorylation by hypothesis-driven multiple-stage mass spectrometry

We describe a strategy, which we term hypothesis-driven multiple-stage mass spectrometry (HMS-MS), for the sensitive detection and identification of phosphopeptides derived from enzymatic digests of phosphoproteins. In this strategy, we postulate that any or all of the potential sites of phosphorylation in a given protein may be phosphorylated. Using this assumption, we calculate the m/z values of all the corresponding singly charged phosphopeptide ions that could, in theory, be produced by the enzyme employed for proteolysis. We test ions at these m/z values for the presence of phosphoserine or phosphothreonine residues using tandem mass spectrometry (MS(2)) in a vacuum MALDI ion trap mass spectrometer, where the neutral loss of the elements of H(3)PO(4) (98 Da) provides a sensitive assay for the presence of phosphopeptides. Subsequent MS(3) analysis of the (M + H - 98)(+) peaks allows us to confirm or reject the hypotheses that the putative phosphopeptides are present in the sample. HMS-MS was successfully applied to the detection and identification of phosphopeptides from substrates of the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28, phosphorylated in vitro (Ipl1) and in vivo (Orc6), basing hypothesis formation on the minimal Cdk consensus phosphorylation motif Ser/Thr-Pro. The method was also used to find in vitro phosphopeptides from a domain of the Drosophila melanogaster protein PERIOD, hypothesizing possible phosphorylations of all Ser/Thr residues without assuming a consensus motif. Our results demonstrate that HMS-MS is a sensitive, highly specific tool for systematically surveying proteins for Ser/Thr phosphorylation, and represents a significant step toward our goal of comprehensive phosphorylation mapping.     

Use of capillary electrophoresis and endogenous fluorescent substrate to monitor intracellular activation of protein kinase A

Here we demonstrate for the first time the use of an endogenous multiphosphorylatable substrate for monitoring the intracellular activation of kinase with capillary electrophoresis. First, we devised a novel PCR-based strategy for controlled generation of short multirepeat DNA sequences and applied this method to generate a green fluorescence protein (GFP)-tagged protein substrate containing eight phosphorylation sites for protein kinase A (PKA). The protein substrate was transiently expressed in C2C12 rat myoblast cells, and intracellular PKA was then activated by adding [8]-bromo-cyclic AMP to the cell culture medium. Phosphorylated product and nonphosphorylated substrate present in the crude cell extract were separated by capillary zone electrophoresis and detected with laser-induced fluorescence of the GFP tag. The identities of two electrophoretic peaks were confirmed by both phosphorylation of the substrate and dephosphorylation of the product in vitro. The proposed method was applied to monitoring the activation of PKA in single myoblast cells. It advantageously allowed us to avoid microinjection of the substrate, the procedure that is both hard to perform and excessively invasive when applied to small mammalian cells.     

Detection of low-abundance protein phosphorylation by selective (18)o labeling and mass spectrometry

Reversible phosphorylation regulates the majority of intracellular networking and pathways. The study of this widely explored post-translational modification is usually challenged by low stoichiometric levels of modification. Many approaches have been developed to overcome this problem and to achieve rigorous characterization of protein phosphorylation. We describe a method for enhanced detection of low-abundance protein phosphorylation that uses selective introduction of (18)O label into phosphorylation sites with H(2)(18)O and mass spectrometric detection. The method was applied to introduce (18)O label into bacterially expressed Aurora A kinase phosphorylation sites and resulted in the representation of phosphorylated peptides as doublets or triplets according to the number of phosphate groups. A total of 28 phosphopeptides were observed by this method.     

Targeted online liquid chromatography electron capture dissociation mass spectrometry for the localization of sites of in vivo phosphorylation in human Sprouty2

We demonstrate a strategy employing collision-induced dissociation for phosphopeptide discovery, followed by targeted electron capture dissociation (ECD) for site localization. The high mass accuracy and low background noise of the ECD mass spectra allow facile sequencing of coeluting isobaric phosphopeptides, with up to two isobaric phosphopeptides sequenced from a single mass spectrum. In contrast to the previously described neutral loss dependent ECD method, targeted ECD allows analysis of both phosphotyrosine peptides and lower abundance phosphopeptides. The approach was applied to phosphorylation analysis of human Sprouty2, a regulator of receptor tyrosine kinase signaling. Fifteen sites of phosphorylation were identified, 11 of which are novel.     

Unleashing Cancer Cells on Surfaces Exposing Motogenic IGDQ Peptides

Thiolated peptides bearing the Ile‐Gly‐Asp (IGD) motif, a highly conserved sequence of fibronectin, are used for the preparation of anisotropic self‐assembled monolayers (SAM gradients) to study the whole‐population migratory behavior of metastatic breast cancer cells (MDA‐MB‐231 cells). Ile‐Gly‐Asp‐Gln‐(IGDQ)‐exposing SAMs sustain the adhesion of MDA‐MB‐231 cells by triggering focal adhesion kinase phosphorylation, similarly to the analogous Gly‐Arg‐Gly‐Asp‐(GRGD)‐terminating surfaces. However, the biological responses of different cell lines interfaced with the SAM gradients show that only those exposing the IGDQ sequence induce significant migration of MDA‐MB‐231 cells. In particular, the observed migratory behavior suggests the presence of cell subpopulations associated with a “stationary” or a “migratory” phenotype, the latter determining a considerable cell migration at the sub‐cm length scale. These findings are of great importance as they suggest for the first time an active role of biological surfaces exposing the IGD motif in the multicomponent orchestration of cellular signaling involved in the metastatic progression.     

Analysis of CheA histidine phosphorylation and its influence on protein stability by high-resolution element and electrospray mass spectrometry

A combination of electrospray mass spectrometry (ESI-MS) and element mass spectrometry (ICPMS) with phosphorus detection was used to characterize histidine phosphorylation (His-48) of the chemotaxis protein CheA quantitatively. The phosphorylation at His-48 was found to be responsible for a stabilization of the protein. For this investigation, the acceptor domain and the kinase domain of the bacterial chemotaxis protein CheA were recombinantly expressed as single proteins. Using in vitro kinase assay conditions, the acceptor domain CheA-H was phosphorylated by the kinase domain CheA-C. The degree of histidine phosphorylation was determined by nanoelectrospray mass spectrometry of intact CheA-H, and was found to be limited to a maximum value of approximately 50%. The site specificity of CheA-H phosphorylation was controlled by nanoESI-MS/MS of the [M + 16H](16+) ion of intact (pHis)-CheA-H and allowed localization of the pHis residue to the region between residues 32 and 86, containing candidates His-48 and His-67, for which His-48 phosphorylation has been described. Analysis of the tryptic digest of in vitro histidine-phosphorylated CheA-H by capillary chromatography coupled to ESI-MS and to ICPMS with phosphorus detection revealed a truncated (pHis)-CheA-H protein as the only phosphorus-containing analyte. Since the truncated (pHis)-CheA-H in the digest was found to exhibit a higher degree of phosphorylation than could be generated by in vitro phosphorylation without trypsin treatment, it is concluded that histidine phosphorylation at His-48 strongly interferes with structural properties of the CheA-H domain in particular with respect to proteolytic degradation by trypsin.     

Protein phosphorylation degree: determination by capillary liquid chromatography and inductively coupled plasma mass spectrometry

Capillary liquid chromatography (muLC) interfaced to inductively coupled plasma mass spectrometry (ICPMS) is introduced as a new micromethod to determine the phosphorylation degree in phosphoproteins and phosphopeptides containing cysteine and/or methionine residues. The stoichiometric phosphorus to sulfur (31P to 32S) ratio is experimentally determined by muLC-ICPMS and converted into the degree of phosphorylation using protein/ peptide sequence information. The method is applied to the phosphoproteins beta-casein, beta-casein, and recombinant protein kinase A catalytic subunit and to synthetic phosphopeptides. The accurate data obtained by muLC-ICPMS allow quantitative assessment of the compound-specific discrimination of the electrospray ionization process between nonphosphorylated and phosphorylated proteins and peptides.     

Analysis of protein phosphorylation by capillary liquid chromatography coupled to element mass spectrometry with 31P detection and to electrospray mass spectrometry

A method for phosphopeptide identification by capillary liquid chromatography (muLC) interfaced alternatively to element mass spectrometry (inductively coupled plasma mass spectrometry, ICPMS) and to electrospray ionization mass spectrometry (ESI-MS) is described. ICPMS is used for 31P detection and ESI-MS provides the corresponding molecular weight information. Alignment of the two separate muLC runs is performed using the baseline distortion at the elution front, which shows up in both muLC-ICPMS and muLC-ESI-MS. Both a quadrupole and a magnetic sector field mass analyzer were used in combination with ICP. The detection limit achieved for the muLC-ICP-HRMS runs is approximately 0.1 pmol of phosphopeptide injected. Without any further precautions, contamination by phosphate-containing compounds at this level was found to be uncritical. The method is demonstrated for the analysis of a complex mixture of synthetic phosphopeptides and a set of tryptic digests of three phosphoproteins. These include beta-casein, activated human MAP kinase ERK1, and protein kinase A catalytic subunit. The tryptic phosphopeptides of these proteins could all be detected and identified by our new strategy. Analysis of three fractions of protein kinase A catalytic subunit with different phosphorylation status gives direct access to the order in which the phosphorylation of the four phosphorylation sites occurs. The two most important aspects of using muLC-ICPMS with 31P detection for phosphopeptide identification are (i) that a high selectivity is achieved and (ii) that the signal intensity is independent of the chemical form of phosphorus and directly proportional to the molar amount of 31P in the muLC eluate. Thus, muLC-ICPMS with 31P detection is introduced as a new, robust, and specific method in phosphoproteomics.