Determination of protein phosphorylation sites by mass spectrometry: a novel electrospray-based method

Several methods are used to identify protein phosphorylation sites. We report a novel electrospray-based method for the determination of phosphorylation sites by mass spectrometry, using two different declustering potential values. This method allows one to obtain, with a single liquid chromatography/mass spectrometry (LC/MS) run, the pattern with either the phosphorylated or the unphosphorylated species of a protein tryptic digest, that can be further analyzed by tracing back the origin of each HPO3-deprived form using the capabilities of tandem mass spectrometers.     

Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry

Quantification of the stoichiometry of phosphorylation is usually achieved using a mixture of phosphatase treatment and differential isotopic labeling. Here, we introduce a new approach to the concomitant determination of absolute protein concentration and the stoichiometry of phosphorylation at predefined sites. The method exploits QconCAT to quantify levels of phosphorylated and nonphosphorylated peptide sequences in a phosphoprotein. The nonphosphorylated sequence is used to determine the absolute protein quantity and serves as a reference to calculate the extent of phosphorylation at the second peptide. Thus, the stoichiometry of phosphorylation and the absolute protein concentration can be determined accurately in a single experiment.     

Analysis of non-covalent protein complexes by capillary electrophoresis--time-of-flight mass spectrometry

A capillary electrophoresis-electrospray ionisation time-of-flight mass spectrometry (CE-ESI-TOF-MS) method for characterisation of non-covalent protein complexes is described using a coaxial liquid sheath-flow sprayer. The CE capillary was connected to the mass spectrometer using a commercial CE-MS sprayer mounted on a ceramic holder of the ESI interface of the mass spectrometer. Using myoglobin (Mb) as an example of non-covalent protein complex, the effect on complex stability caused by organic modifiers added to the sheath liquid was analysed. Depending on the amount of methanol, either intact Mb or the apoprotein and the prosthetic heme group were detected.     

应用双向电泳和质谱联用技术研究乳源蛋白的差异性

应用双向电泳和质谱联用技术,对不同乳源蛋白的差异性进行了研究。根据ImageMaster 2DPlatinum图像分析软件对不同乳源酪蛋白和乳清蛋白的双向电泳(2-DE)图谱进行蛋白斑点的匹配分析,获得21个存在于水牛奶中主要分布在低丰度蛋白区的酪蛋白差异蛋白点和24个存在于水牛奶中乳清蛋白差异蛋白点。这些差异蛋白点经质谱鉴定分析,得到4个属于水牛奶酪蛋白的主要组分和2个与水牛奶中酪蛋白有较高同源性的新组分,同时获得4个属于水牛奶乳清蛋白的主要组分和3个与水牛奶中乳清蛋白有较高同源性的组分。     

The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites

Protein phosphorylation analysis is an enormous challenge. This review summarises the currently used techniques, which are based on radiolabelling and mass spectrometry as well as electrophoretic and chromatographic separation. Many methods exist, but there is still no single procedure applicable to all phosphoproteins. MS is able to deliver information about the location of phosphorylation sites, but phosphospecific properties with respect to ionisation present obstacles. Therefore, multidimensional approaches involving several analytical methods are often necessary to conquer phosphorylation site identification.Abbreviations 2D Two-dimensional - CE Capillary electrophoresis - CID Collision-induced dissociation - ECD Electron capture dissociation - ESI Electrospray ionisation - FT-ICR Fourier transform ion cyclotron resonance - HPLC High performance liquid chromatography - ICAT Isotope coded affinity tags - ICP Inductively-coupled plasma - IDA Immino-diacetic acid - IMAC Immobilised metal affinity chromatography - IRMPD Infrared multiphoton dissociation - IT Ion trap - MALDI Matrix-assisted laser desorption/ionisation - MRP14 Myeloid-related protein 14 - MS Mass spectrometry - NTA Nitrilo-triacetic acid - PAGE Polyacrylamide gel electrophoresis - PDI Protein disulfide isomerase - pS Phosphoserine residue - PSD Post-source decay - pT Phosphothreonine residue - PVDF Polyvinylidene fluoride - pY Phosphotyrosine residue - Q-TOF Quadrupole-time-of-flight - RP Reversed phase - SIM Single-ion monitoring - SDS Sodium dodecyl sulfate - SORI Sustained off-resonance irradiation - TLC Thin-layer chromatography - TOF Time-of-flight An erratum to this article can be found at     

Investigating quantitation of phosphorylation using MALDI-TOF mass spectrometry

Despite advances in methods and instrumentation for analysis of phosphopeptides using mass spectrometry, it is still difficult to quantify the extent of phosphorylation of a substrate because of physiochemical differences between unphosphorylated and phosphorylated peptides. Here we report experiments to investigate those differences using MALDI-TOF mass spectrometry for a set of synthetic peptides by creating calibration curves of known input ratios of peptides/phosphopeptides and analyzing their resulting signal intensity ratios. These calibration curves reveal subtleties in sequence-dependent differences for relative desorption/ionization efficiencies that cannot be seen from single-point calibrations. We found that the behaviors were reproducible with a variability of 5-10% for observed phosphopeptide signal. Although these data allow us to begin addressing the issues related to modeling these properties and predicting relative signal strengths for other peptide sequences, it is clear that this behavior is highly complex and needs to be further explored.     

Honey protein extraction and determination by mass spectrometry

There are relatively limited studies on the protein of honey samples mainly because of the low amount of protein in honey (0.1–0.5 %), the difficulty in extracting honey protein from the sugar-rich environment, and the hindrance of protein characterization by conventional approaches. Several protein extraction methods such as mechanical (ultrafiltration and ultracentrifugation) and chemical (precipitation) techniques have been applied to different types of honey samples. Most of these studies reported the quantity and molecular size of honey protein from gel electrophoresis, but were unable to identify and characterize the protein. This limitation might be due to the low capacity of analytical equipment in those days. Although different precipitants have also been used, not all them are compatible with mass spectrometric methods during downstream analysis. As a result, the sample preparation step is essential in order to confidently characterize the low and varied amount of honey protein. Nowadays, honey protein is getting attention from researchers because of its potential activity in pharmacological applications. Therefore, honey protein extraction and determination by mass spectrometry are critically reviewed in order to stimulate further honey protein research.

Determination of protein phosphorylation by extracellular signal-regulated kinase using capillary electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Extracellular signal-regulated kinase (ERK) is a key regulatory enzyme mediating cell responses to mitogenic stimulation and is one of the key components in linking growth factor receptor activation to serine/threonine protein phosphorylation processes. Phosphorylation reaction by ERK plays an important role in many signal transduction pathways. ERK phosphorylates numerous substrates such as MBP, microtubule-associated protein 2 (MAP2) and nuclear protein. In particular, MBP is a substrate commonly employed for the detection of ERK activity and contains the consensus primary sequence PRT97P. In this paper, we compared the degree of the phosphorylation reaction of MBP substrate peptides by ERK with the three different MBP substrate peptides, MBP1(KNIVTPRTPPPSQGK), MBP2(VPRTPGGRR) and MBP3(APRTPGGRR) in order to select an efficient substrate peptide for phosphorylation reaction by ERK. The results showed that the MBP3 peptide is the most efficient substrate for phosphorylation reaction by ERK. Using MBP3 peptide, the phosphorylation reaction of MBP by ERK was monitored with both matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis (CE). Our results demonstrate the feasibility of the CE method, the method being a simple and reliable technique in determining and characterizing various kinds of enzyme reaction especially including kinase enzymes.     

Analysis of protein interaction networks using mass spectrometry compatible techniques

The ability to map protein-protein interactions has grown tremendously over the last few years, making it possible to envision the mapping of whole or targeted protein interaction networks and to elucidate their temporal dynamics. The use of mass spectrometry for the study of protein complexes has proven to be an invaluable tool due to its ability to unambiguously identify proteins from a variety of biological samples. Furthermore, when affinity purification is combined with mass spectrometry analysis, the identification of multimeric protein complexes is greatly facilitated. Here, we review recent developments for the analysis of protein interaction networks by mass spectrometry and discuss the integration of different bioinformatic tools for predicting, validating, and managing interaction datasets.     

Analysis of protein complexes with hydrogen exchange and mass spectrometry

Analysis of protein complexes using hydrogen exchange (HX) combined with high resolution electrospray mass spectrometry (MS) is demonstrated. HX MS offers the possibility to analyze the strength of binding in protein complexes, to identify regions that undergo binding induced structural changes, and to study the nature (hydrophobic, electrostatic, etc.) of binding between two or more proteins. In the current work, a heteromeric complex containing UBC9 (an E2 conjugating enzyme) and SUMO-1 (a ubiquitin-like modifier) was investigated by incubating the complex in D2O and measuring the amount of deuterium incorporation with MS. SUMO-1 had significant changes in deuterium levels when bound to UBC9. In contract, few or no changes in deuterium levels were detected in UBC9 when part of the complex, even at the binding interface. Titrations were used to estimate the binding constant for the complex. The nature of the interface was probed by creating a site-directed mutant form of UBC9. The mutant form showed no detectable binding to SUMO-1 and thereby suggested that binding between these two proteins is primarily electrostatically driven. This application of HX MS demonstrates its value in the study of protein complexes and protein machinery.     

Analysis of sulfobutyl ether-beta-cyclodextrin mixtures by ion-spray mass spectrometry and liquid chromatography-ion-spray mass spectrometry

A comparison is made of the retention properties of additives applied as positively charged pseudo-stationary phases for electrokinetic chromatography of neutral analytes. All additives have a quaternary ammonium as functional group. The polymeric additive [poly(N,N,N',N'-tetramethyl-N-trimethylenehexamethylenediammonium), Polybrene] has a concentration of 2% (w/w) in the background electrolyte (acetate, pH 5.2). Monomeric octyltrimethylammonium (OTMA) was used at a concentration below or above its critical micelle concentration (CMC) (140 mmol/l). At a concentration (259 mmol/l) above the CMC the system is that normally used for micellar electrokinetic chromatography with cationic micelles. However, even below the CMC, where OTMA is present as monomer, retention of the neutral analytes is observed as well. In all systems coating of the capillary wall with Polybrene establishes an electroosmotic flow directed towards the anode, counter-migrating to the electrophoretic movement of the additive. Based on the measurement of the mobility of the analytes (15 small, monofunctional aromatic compounds with different functional groups), their capacity factors, k(i), were determined in all systems. Low correlation of the k(i) values is observed between the particular systems, indicating their different selectivity at least for individual pairs of analytes. Based on the log k(i) values, a linear free energy relationship was applied to elucidate the main types of chemical interaction responsible for retention. As a result, cavity formation and n or pi electron interactions were found being significant for the micellar OTMA system, which agrees with findings described in the literature for other (cationic and anionic) micellar systems. For the polymeric system and for the monomeric OTMA system, the significant retention parameter is indicating n and pi electron interactions.     

Identification of phosphorylation sites in proteins by nanospray quadrupole ion trap mass spectrometry

A method is described for identifying serine phosphorylation sites in proteins, based on conventional (32)P labeling followed by electrophoretic separation, 'in-gel' digestion with a protease, peptide extraction, reversed-phase high-performance liquid chromatographic separation and collection and off-line analysis of the radioactive fractions by nanospray ion trap mass spectrometry. The method was successfully applied to the identification of three phosphorylation sites in two proteins which were subjected to in vitro phosphorylation under physiological conditions. Different combinations of the various scanning modes of the ion trap, including high-resolution, multiple subfragmentation (or MS(n)) and fast scan analysis, were employed to identify the phosphopeptides, determine their sequence and localize the exact site of phosphorylation. 'Blind' fragmentation using fast scans was used to analyze a phosphopeptide which was undetectable in other scanning modes. The sequence, phosphorylation site and double cysteine modification of the potassium adduct of a peptide containing 35 residues were also determined by multiple fragmentation. The results not only support the validity of the proposed method for routine identification of phosphorylation sites, but also demonstrate the exceptional capability of off-line ion trap mass spectrometry in combination with nanospray ionization for performing very detailed studies on the structure of peptides.     

Analysis of intact erythropoietin and novel erythropoiesis-stimulating protein by capillary electrophoresis-electrospray-ion trap mass spectrometry

Recombinant human erythropoietin (rHuEPO) and novel erythropoiesis-stimulating protein (NESP) were analyzed by CE-ESI-MS using an IT as analyzer. The IT parameters were optimized by direct infusion of solutions of different intact proteins (myoglobin, transferrin, alpha1-acid glycoprotein and fetuin) with different degrees of glycosylation (from 0 to 35% w/w). Two physically adsorbed capillary coatings from UltraTol Pre-Coats (low normal (LN) and high reverse (HR)) were evaluated for the separation of rHuEPO and NESP glycoforms by CE-ESI-IT-MS. The results obtained with the neutral LN coating suggest that an IT mass spectrometer enables identification of the main glycoforms of a complex glycoprotein such as rHuEPO. Although LN provided acceptable glycoform resolution for rHuEPO, the separation obtained for NESP was less significant due to the higher microheterogeneity of this glycoprotein. Reproducibility studies confirmed the lack of stability and bleeding of the LN coating, which caused problems with MS detection, such as a dramatic loss of sensitivity and the presence of peaks in the mass spectra corresponding to molecular ions in the coating. In contrast, the cationic HR coating gave faster but poorer glycoform separations due to the presence of an anodal EOF. However, the positive charge of the coating provided enhanced hydrolytic stability, making it more suitable than the LN coating for the on-line MS coupling.     

Analysis of quaternary protein ensembles by matrix assisted laser desorption/ionization mass spectrometry

The intact noncovalent structure of the homo-oligomeric complexes of streptavidin (52 kDa), alcohol dehydrogenase (150 kDa), and beef liver catalase (240 kDa) have been observed using the matrix 2,6-dihydroxyacetophenone in an organic solvent. Intact streptavidin tetramers could also be observed with ferulic acid and other hydroxyacetophenone derivatives. Intact complexes are observed only for the first shot at a given position, which may be due to physical segregation or precipitation of the noncovalent complexes at the crystal surface. This effect is independent of the macroscopic crystal structure or the type of substrate (hydrophobic versus hydrophilic). Observation of intact complexes is not affected by addition of less than 10 mM salts or buffers, and appears to be independent of the pH stability range of the protein samples investigated.