Phosphoproteome mapping of cardiomyocyte mitochondria in a rat model of heart failure

Mitochondria are complex organelles essential to cardiomyocyte survival. Protein phosphorylation is emerging as a key regulator of mitochondrial function. In the study reported here, we analyzed subsarcolemmal (SSM) mitochondria harvested from rats who have received 4 weeks of aldosterone/salt treatment to simulate the neurohormonal profile of human congestive heart failure. Our objective was to obtain an initial qualitative inventory of the phosphoproteins in this biologic system. SSM mitochondria were harvested, and the phosphoproteome was analyzed with a gel-free bioanalytical platform. Mitochondrial proteins were digested with trypsin, and the digests were enriched for phosphopeptides with immobilized metal ion affinity chromatography. The phosphopeptides were analyzed by ion trap liquid chromatography–tandem mass spectrometry, and the phosphoproteins identified via database searches. Based on MS/MS and MS³ data, we characterized a set of 42 phosphopeptides that encompassed 39 phosphorylation sites. These peptides mapped to 26 proteins, for example, long-chain specific acyl-CoA dehydrogenase, Complex III subunit 6, and mitochondrial import receptor TOM70. Collectively, the characterized phosphoproteins belong to diverse functional modules, including bioenergetic pathways, protein import machinery, and calcium handling. The phosphoprotein panel discovered in this study provides a foundation for future differential phosphoproteome profiling toward an integrated understanding of the role of mitochondrial phosphorylation in heart failure.     

Large-scale phosphoproteomics analysis of whole saliva reveals a distinct phosphorylation pattern

In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique phosphopeptides sites were identified representing 85 distinct phosphoproteins at 2.3% global FDR. From these peptides, 129 distinct phosphorylation sites were identified of which 57 were previously known, but only 11 of which had been previously identified in whole saliva. Cellular localization analysis revealed salivary phosphoproteins had a distribution similar to all known salivary proteins, but with less relative representation in "extracellular" and "plasma membrane" categories compared to salivary glycoproteins. Sequence alignment showed that phosphorylation occurred at acidic-directed kinase, proline-directed, and basophilic motifs. This differs from plasma phosphoproteins, which predominantly occur at Golgi casein kinase recognized sequences. Collectively, these results suggest diverse functions for salivary phosphoproteins and multiple kinases involved in their processing and secretion. In all, this study should lay groundwork for future elucidation of the functions of salivary protein phosphorylation.     

Nanoliter sample handling combined with microspot MALDI-MS for detection of gel-separated phosphoproteins

We describe a microspot matrix-assisted laser desorption ionization (MALDI) mass spectrometric approach to analyze gel-separated phosphoproteins. This method involves in-gel digestion of phosphoproteins after gel separation, followed by open tubular capillary (OTC) immobilized metal-ion affinity chromatography (IMAC) to capture the phosphopeptides with markedly reduced interferences from nonphosphorylated peptides. Nanoliter-volume of ammonium phosphate is used to elute the phosphopeptides captured on the capillary tube. After mixing with a small volume of matrix solution in the capillary, the effluent is deposited in a microspot on a sample plate for MALDI-MS analysis. It is also shown that, with peptide esterification after in-gel digestion of a phosphoprotein, negative ion detection in MALDI gives a distinct advantage over the positive ion mode of operation for phosphopeptide analysis, even without IMAC enrichment. However, the OTC-IMAC technique is demonstrated to be superior to the approach of negative ion detection of esterified in-gel digests without IMAC. OTC-IMAC is found to be sufficiently selective to capture phosphopeptides from in-gel digest of a gel band containing predominately one protein and the combination of peptide esterification and IMAC enrichment does not provide any real advantage. Using a standard phosphoprotein alpha-casein as a model system, we demonstrate that this OTC-IMAC method can detect a number of phosphopeptides after in-gel digestion with mid-fmol protein sample loading. An example of real world applications of this method is illustrated in the characterization of a fusion protein, His182, expressed in E. coli.     

Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane Cl- channel regulated by protein phosphorylation. To identify cAMP-dependent protein kinase (PKA)-phosphorylated residues in full-length CFTR, immobilized metal-ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe3+) nitrilotriacetic (NTA). Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA-phosphorylated recombinant NBD1-R protein from CFTR. Fe(3+)-loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly-His-containing peptides were co-purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA-phosphorylated full-length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and collision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660Ser, 737Ser, and 795Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700Ser, 712Ser, 768Ser, and 813Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753Ser in full-length CFTR.     

Phosphoproteomic analysis of chromoplasts from sweet orange during fruit ripening

Like other types of plastids, chromoplasts have essential biosynthetic and metabolic activities which may be regulated via post‐translational modifications, such as phosphorylation, of their resident proteins. We here report a proteome‐wide mapping of in vivo phosphorylation sites in chromoplast‐enriched samples prepared from sweet orange [Citrus sinensis (L.) Osbeck] at different ripening stages by titanium dioxide‐based affinity chromatography for phosphoprotein enrichment with LC‐MS/MS. A total of 109 plastid‐localized phosphoprotein candidates were identified that correspond to 179 unique phosphorylation sites in 135 phosphopeptides. On the basis of Motif‐X analysis, two distinct types of phosphorylation sites, one as proline‐directed phosphorylation motif and the other as casein kinase II motif, can be generalized from these identified phosphopeptides. While most identified phosphoproteins show high homology to those already identified in plastids, approximately 22% of them are novel based on BLAST search using the public databases PhosPhAt and P³DB. A close comparative analysis showed that approximately 50% of the phosphoproteins identified in citrus chromoplasts find obvious counterparts in the chloroplast phosphoproteome, suggesting a rather high‐level of conservation in basic metabolic activities in these two types of plastids. Not surprisingly, the phosphoproteome of citrus chromoplasts is also characterized by the lack of phosphoproteins involved in photosynthesis and by the presence of more phosphoproteins implicated in stress/redox responses. This study presents the first comprehensive phosphoproteomic analysis of chromoplasts and may help to understand how phosphorylation regulates differentiation of citrus chromoplasts during fruit ripening.     

Rapid enrichment of phosphopeptides and phosphoproteins from complex samples using magnetic particles coated with alumina as the concentrating probes for MALDI MS analysis

In this study, we used nanocomposite magnetic particles coated with alumina as the affinity probes to selectively concentrate phosphorylated peptides and proteins from a low volume of sample solution. Tryptic digest products of phosphoproteins including alpha and beta-caseins, human protein phosphatase inhibitor 1, nonfat milk, egg white, and a cell lysate were used as the samples to demonstrate the feasibility of this approach. In only 30 and 90 s, phosphopeptides and phosphoproteins sufficient for characterization by MALDI-MS were enriched by the particles, respectively. Proteins trapped on the particles could be directly digested on the particles. The same particles in the digest solution were employed for enrichment of phosphopeptides. We estimated the required time for performing the enrichment of phosphopeptides from complex samples and characterization by MALDI MS was within 5 min. A small volume (50 microL) and a low concentration (5 x 10(-10) M) of tryptic digest product of a phosphoprotein sample could be dramatically enriched and characterized using this approach.     

Characterization of the phosphoproteome in androgen-repressed human prostate cancer cells by Fourier transform ion cyclotron resonance mass spectrometry

Androgen-repressed human prostate cancer, ARCaP, grows and is highly metastatic to bone and soft tissues in castrated mice. The molecular mechanisms underlying the aberrant responses to androgen are not fully understood. Here, we apply state-of-the-art mass spectrometry methods to investigate the phosphoproteome profiles in ARCaP cells. Because protein biological phosphorylation is always substoichiometric and the ionization efficiency of phosphopeptides is low, selective enrichment of phosphorylated proteins/peptides is required for mass spectrometric analysis of phosphorylation from complex biological samples. Therefore, we compare the sensitivity, efficiency, and specificity for three established enrichment strategies: calcium phosphate precipitation (CPP), immobilized metal ion affinity chromatography (IMAC), and TiO(2)-modified metal oxide chromatography. Calcium phosphate precipitation coupled with the TiO(2) approach offers the best strategy to characterize phosphorylation in ARCaP cells. We analyzed phosphopeptides from ARCaP cells by LC-MS/MS with a hybrid LTQ/FT-ICR mass spectrometer. After database search and stringent filtering, we identified 385 phosphoproteins with an average peptide mass error of 0.32 ± 0.6 ppm. Key identified oncogenic pathways include the mammalian target of rapamycin (mTOR) pathway and the E2F signaling pathway. Androgen-induced proliferation inhibitor (APRIN) was detected in its phosphorylated form, implicating a molecular mechanism underlying the ARCaP phenotype.     

MALDI‐TOF and nESI Orbitrap MS/MS identify orthogonal parts of the phosphoproteome

Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows. In this study, we compared tandem mass spectrometry (MS/MS) on matrix assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) and nano‐electrospray ionization (nESI) Orbitrap instruments with respect to their ability to identify phosphopeptides from complex proteome digests. Phosphopeptides were enriched from tryptic digests of cell lines using Fe‐IMAC column chromatography and subjected to LC‐MS/MS analysis. We found that the two analytical workflows exhibited considerable orthogonality. For instance, MALDI‐TOF MS/MS favored the identification of phosphopeptides encompassing clear motif signatures for acidic residue directed kinases. The extent of orthogonality of the two LC‐MS/MS systems was comparable to that of using alternative proteases such as Asp‐N, Arg‐C, chymotrypsin, Glu‐C and Lys‐C on just one LC‐MS/MS instrument. Notably, MALDI‐TOF MS/MS identified an unexpectedly high number and percentage of phosphotyrosine sites (～20% of all sites), possibly as a direct consequence of more efficient ionization. The data clearly show that LC‐MALDI MS/MS can be a useful complement to LC‐nESI MS/MS for phosphoproteome mapping and particularly so for acidic and phosphotyrosine containing peptides.     

Mitochondrial phosphoproteome revealed by an improved IMAC method and MS/MS/MS

IMAC in combination with mass spectrometry is a promising approach for global analysis of protein phosphorylation. Nevertheless this approach suffers from two shortcomings: inadequate efficiency of IMAC and poor fragmentation of phosphopeptides in the mass spectrometer. Here we report optimization of the IMAC procedure using (32)P-labeled tryptic peptides and development of MS/MS/MS (MS3) for identifying phosphopeptide sequences and phosphorylation sites. The improved IMAC method allowed recovery of phosphorylated tryptic peptides up to approximately 77% with only minor retention of unphosphorylated peptides. MS3 led to efficient fragmentation of the peptide backbone in phosphopeptides for sequence assignment. Proteomics of mitochondrial phosphoproteins using the resulting IMAC protocol and MS3 revealed 84 phosphorylation sites in 62 proteins, most of which have not been reported before. These results revealed diverse phosphorylation pathways involved in the regulation of mitochondrial functions. Integration of the optimized batchwise IMAC protocol with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation.     

Laser-induced dissociation of phosphorylated peptides using matrix assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Reversible phosphorylation is one of the most important posttranslational modifications of cellular proteins. Mass spectrometry is a widely used technique in the characterization of phosphorylated proteins and peptides. Similar to nonmodified peptides, sequence information for phosphopeptides digested from proteins can be obtained by tandem mass analysis using either electrospray ionization or matrix assisted laser desorption/ionization (MALDI) mass spectrometry. However, the facile loss of neutral phosphoric acid (H₃PO₄) or HPO₃ from precursor ions and fragment ions hampers the precise determination of phosphorylation site, particularly if more than one potential phosphorylation site or concensus sequence is present in a given tryptic peptide. Here, we investigated the fragmentation of phosphorylated peptides under laser-induced dissociation (LID) using a MALDI-time-of-flight mass spectrometer with a curved-field reflectron. Our data demonstrated that intact fragments bearing phosphorylated residues were produced from all tested peptides that contain at least one and up to four phosphorylation sites at serine, threonine, or tyrosine residues. In addition, the LID of phosphopeptides derivatized by N-terminal sulfonation yields simplified MS/MS spectra, suggesting the combination of these two types of spectra could provide an effective approach to the characterization of proteins modified by phosphorylation.     

Identification of three previously unknown in vivo protein phosphorylation sites in thylakoid membranes of Arabidopsis thaliana

The proteins in plant photosynthetic thylakoid membranes undergo light-induced phosphorylation, but only a few phosphoproteins have been characterized. To access the unknown sites of in vivo protein phosphorylation the thylakoid membranes were isolated from Arabidopsis thaliana grown in normal light, and the surface-exposed peptides were cleaved from the membranes by trypsin. The peptides were methylated and subjected to immobilized metal affinity chromatography, and the enriched phosphopeptides were sequenced using tandem nanospray quadrupole time-of-flight mass spectrometry. Three new phosphopeptides were revealed in addition to the five known phosphorylation sites in photosystem II proteins. All phosphopeptides are found phosphorylated at threonine residues implementing a strict threonine specificity of the thylakoid kinases. For the first time protein phosphorylation is found in photosystem I. The phosphorylation site is localized to the first threonine in the N terminus of PsaD protein that assists in the electron transfer from photosystem I to ferredoxin. A new phosphorylation site is also revealed in the acetylated N terminus of the minor chlorophyll a-binding protein CP29. The third novel phosphopeptide, composed of 25 amino acids, belongs to a nuclear encoded protein annotated as "expressed protein" in the Arabidopsis database. The protein precursor has a chloroplast-targeting peptide followed by the mature protein with two transmembrane helices and a molecular mass of 14 kDa. This previously uncharacterized protein is named thylakoid membrane phosphoprotein of 14 kDa (TMP14). The finding of the novel phosphoproteins extends involvement of the redox-regulated protein phosphorylation in photosynthetic membranes beyond the photosystem II and its light-harvesting antennae.     

Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry

Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poor specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides. Among the identified sequences, six originated from different isoforms of the plasma membrane H(+)-ATPase and defined two previously unknown phosphorylation sites at the regulatory C terminus. The potential for large-scale identification of phosphorylation sites on plasma membrane proteins will have wide-ranging implications for research in signal transduction, cell-cell communication, and membrane transport processes.     

Concerted experimental approach for sequential mapping of peptides and phosphopeptides using C18-functionalized magnetic nanoparticles

An integrated analytical approach for the enrichment, detection, and sequencing of phosphopeptides using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS) was developed. On the basis of C18-functionalized Fe3O4 nanoparticles, the enrichment method was designed not only to specifically trap phosphopeptides, but also nonphosphorylated peptides, both of which can be subsequently desorbed selectively and directly for MALDI-MS analysis without an elution step. Peptide binding is afforded by the C18-derivatization, whereas the highly selective capture of phosphopeptides is based on higher binding affinity afforded by additional metal chelating interaction between the Fe3O4 nanoparticles and the phosphate groups. Upon binding, the initial aqueous wash allows desalting, while a second and a third wash with high acetonitrile content coupled with diluted sulfuric acid and ammonia removes most of the bound nonphosphorylated peptides. Selective or sequential mapping of the peptides and phosphopeptides can, thus, be effected by spotting the washed nanoparticles onto the MALDI target plate along with judicious choice of matrices. The inclusion of phosphoric acid in a 2,5-dihydroxybenzoic acid matrix allows the desorption and detection of phosphopeptides, whereas an alpha-cyano-4-hydroxy-cinnamic acid matrix with formic acid allows only the desorption of nonphosphorylated peptides. The method used to enrich phosphopeptides prior to MS applications is more sensitive and tolerable to sodium dodecyl sulfate than IMAC. We have demonstrated the applicability of C18-functionalized Fe3O4 nanoparticles in the detection of in vitro phosphorylation sites on the myelin basic protein, and at least 17 phosphopeptides were identified, including one previously uncharacterized site.     

Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry

We present a robust and general method for the identification and relative quantification of phosphorylation sites in complex protein mixtures. It is based on a new chemical derivatization strategy using a dendrimer as a soluble polymer support and tandem mass spectrometry (MS/MS). In a single step, phosphorylated peptides are covalently conjugated to a dendrimer in a reaction catalyzed by carbodiimide and imidazole. Modified phosphopeptides are released from the dendrimer via acid hydrolysis and analyzed by MS/MS. When coupled with an initial antiphosphotyrosine protein immunoprecipitation step and stable-isotope labeling, in a single experiment, we identified all known tyrosine phosphorylation sites within the immunoreceptor tyrosine-based activation motifs (ITAM) of the T-cell receptor (TCR) CD3 chains, and previously unknown phosphorylation sites on total 97 tyrosine phosphoproteins and their interacting partners in human T cells. The dynamic changes in phosphorylation were quantified in these proteins.     

A systematic approach to the analysis of protein phosphorylation

Reversible protein phosphorylation has been known for some time to control a wide range of biological functions and activities. Thus determination of the site(s) of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. However, direct determination of individual phosphorylation sites occurring on phosphoproteins in vivo has been difficult to date, typically requiring the purification to homogeneity of the phosphoprotein of interest before analysis. Thus, there has been a substantial need for a more rapid and general method for the analysis of protein phosphorylation in complex protein mixtures. Here we describe such an approach to protein phosphorylation analysis. It consists of three steps: (1) selective phosphopeptide isolation from a peptide mixture via a sequence of chemical reactions, (2) phosphopeptide analysis by automated liquid chromatography-tandem mass spectrometry (LC-MS/MS), and (3) identification of the phosphoprotein and the phosphorylated residue(s) by correlation of tandem mass spectrometric data with sequence databases. By utilizing various phosphoprotein standards and a whole yeast cell lysate, we demonstrate that the method is equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins, and is capable of selectively isolating and identifying phosphopeptides present in a highly complex peptide mixture.     

Characterization of a TiO₂ enrichment method for label-free quantitative phosphoproteomics

Phosphorylation is a protein post-translational modification with key roles in the regulation of cell biochemistry and signaling. In-depth analysis of phosphorylation using mass spectrometry is permitting the investigation of processes controlled by phosphorylation at the system level. A critical step of these phosphoproteomics methods involves the isolation of phosphorylated peptides from the more abundant unmodified peptides produced by the digestion of cell lysates. Although different techniques to enrich for phosphopeptides have been reported, there are limited data on their suitability for direct quantitative analysis by MS. Here we report a TiO₂ based enrichment method compatible with large-scale and label-free quantitative analysis by LC–MS/MS. Starting with just 500 μg of protein, the technique reproducibly isolated hundreds of peptides, >85% of which were phosphorylated. These results were obtained by using relatively short LC–MS/MS gradient runs (45 min) and without any previous separation step. In order to characterize the performance of the method for quantitative analyses, we employed label-free LC–MS/MS using extracted ion chromatograms as the quantitative readout. After normalization, phosphopeptides were quantified with good precision (coefficient of variation was 20% on average, n = 900 phosphopeptides), linearity (correlation coefficients >0.98) and accuracy (deviations <20%). Thus, phosphopeptide ion signals correlated with the concentration of the respective phosphopeptide in samples, making the approach suitable for in-depth relative quantification of phosphorylation by label-free LC–MS/MS.     

磷蛋白组的研究技术及其进展

真核细胞中蛋白质磷酸化是一个重要事件。真核细胞利用可逆的蛋白磷酸化来控制许多细胞过程包括信号转换、基因表达、细胞周期等。磷蛋白组的研究涉及磷蛋白的分离和鉴定 ,磷酸化残基定位和定量分析。由于蛋白质磷酸化是一个动态过程 ,在细胞中磷蛋白含量低 ,磷酸化位点可变 ,且磷酸肽的质谱信号常常会受到抑制 ,所以磷蛋白的分析存在更多的困难。本文介绍了国内外在磷酸蛋白的分离鉴定及定量分析方面的研究技术以及进展情况。目前 ,质谱仍然是核心的鉴定技术 ,寻找更好富集方法是最大的挑战。定量蛋白组学是对蛋白质的差异表达进行精确的定量分析。目前还不存在一种独立的方法可以完成磷蛋白的分离、鉴定 ,以及磷酸位点的定位和定量分析。随着样品分离技术和相关仪器的发展 ,磷酸蛋白快速、准确、全面分析鉴定将能够实现。     

Comparison of different IMAC techniques used for enrichment of phosphorylated peptides.

Four commercially available immobilized metal ion affinity chromatography (IMAC) methods for phosphopeptide enrichment were compared using small volumes and concentrations of phosphopeptide mixtures with or without extra-added bovine serum albumin (BSA) nonphosphorylated peptides. Addition of abundant tryptic BSA peptides to the phosphopeptide mixture increases the demand for selective IMAC capture. While SwellGel gallium Discs, IPAC Metal Chelating Resin, and ZipTipMC Pipette Tips allow for the possibility of enriching phosphopeptides, the Gyrolab MALDI IMAC1 also presents the possibility of verifying existing phosphopeptides after a dephosphorylation step. Phosphate-containing peptides are identified through a mass shift between phosphorylated and dephosphorylated spectra of 80 Da (or multiples of 80 Da). This verification is useful if the degree of phosphorylation is low in the sample or if the ionization is unfavorable, which often is the case for phosphopeptides. A peptide mixture in which phosphorylated serine, threonine, and tyrosine were represented was diluted in steps and thereafter enriched using the four different IMAC methods prior to analyses with matrix assisted laser desorption/ionization mass spectrometry. The enrichment of phosphopeptides using SwellGel Gallium Discs or Gyrolab MALDI IMAC1 was not significantly affected by the addition of abundant BSA peptides added to the sample mixture, and the achieved detection limits using these techniques were also the lowest. All four of the included phosphopeptides were detected by MALDI-MS only after enrichment using the Gyrolab MALDI IMAC1 compact disc (CD) and detection down to low femtomole levels was possible. Furthermore, selectivity, reproducibility, and detection for a number of other phosphopeptides using the IMAC CD are reported herein. For example, two phosphopeptides sent out in a worldwide survey performed by the Proteomics Research Group (PRG03) of the Association of Biomolecular Resource Facilities (ABRF) were detected and verified by means of the 80 Da mass shift achieved by on-column dephosphorylation.     

The phosphoproteome of a Chlamydomonas reinhardtii eyespot fraction includes key proteins of the light signaling pathway

Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca(2+)-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii.