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.     

Detection of tyrosine phosphorylated peptides via skimmer collision-induced dissociation/ion trap mass spectrometry

Phosphorylation of proteins is an important post-translational protein modification in cellular response to environmental change and occurs in both prokaryotes and eukaryotes. Identification of the amino acid on individual proteins that become phosphorylated in response to extracellular stimulus is essential for understanding the mechanisms involved in the intracellular signals that these modifications facilitate. Most protein kinases catalyze the phosphorylation of proteins on serine, threonine or tyrosine. Although tyrosine phosphorylation is often the least abundant of the three major phosphorylation sites, it is important owing to its role in signal pathways. Currently available methods for the identification of phosphorylation sites can often miss low levels of tyrosine phosphorylations. This paper describes a method for the identification of phosphotyrosine-containing peptides using electrospray ionization on an ion trap mass spectrometer. Skimmer-activated collision-induced dissociation (CID) was used to generate the phosphotyrosine immonium ion at m/z 216. This method is gentle enough that the protonated molecule of the intact peptide is still observed. In-trap CID was employed for the verification of the phosphotyrosine immonium ion. Using this technique, low levels of phosphotyrosine-containing peptides can be identified from peptide mixtures separated by nanoflow micro liquid chromatography/mass spectrometry.     

Mapping <Emphasis Type="Italic">N</Emphasis>-terminus phosphorylation sites and quantitation by stable isotope dimethyl labeling

We have previously coupled stable isotope dimethyl labeling with IMAC enrichment for quantifying the extent of protein phosphorylation in vivo. The enhanced a₁ signal of dimethylated peptides served as a unique mass tag for unequivocal identification of the N-terminal amino acids. In this study, we demonstrate that the a₁ ion could further assist in mapping the precise phosphorylation site near the N-terminal region and allow the determination of the exact site and level of phosphorylation in one step by stable isotope dimethyl labeling. We show that the a₁ ion signal was suppressed for dimethylated peptides with a phosphorylation site at the N-terminus Ser/Thr residue (N-p*Ser/Thr) but was still enhanced for N-terminus Tyr residue (N-p*Tyr) or internal Ser/Thr residues (-p*Ser/Thr). Based on the dominant de-phosphorylated molecular ions and b-H₃PO₄ ions for N-p*Ser/Thr, we propose that dimethyl labeling increases the basicity of the N-terminus and accelerates the de-phosphorylation for N-p*Ser/Thr precursors, which, however, suppresses the a₁ ion enhancement due to the resulting unsaturated covalent bond on C _α of the N-terminus amino acid. Using this method, we excluded three Ser/Thr phosphorylation sites in A431 cells, two of which, however, were previously reported to be phosphorylation sites; we confirmed three known phosphorylation sites in A431 cells and quantified their ratios upon EGF treatment. Notably, we identified a novel phosphorylation site on Ser43 residue at N-terminus of the tryptic peptide derived from SVH protein in pregnant rat uteri. SVH protein has not been reported or implied with any phosphorylation event, and our data show that the Ser43 of SVH is an intrinsic phosphorylation site in pregnant rat uteri and that its phosphorylation level was slightly decreased upon c-AMP treatment.     

Analysis of protein phosphorylation by mass spectrometry

Phosphorylation is one of the most frequently occurring post-translational modifications in proteins. In eukaryotic cells, protein phosphorylation on serine, threonine and tyrosine residues plays a crucial role as a modulator of protein function. A comprehensive analysis of protein phosphorylation involves the identification of the phosphoproteins, the exact localization of the residues that are phosphorylated and the quantitation of phosphorylation. In this short review we will summarize and discuss the methodologies currently available for the analysis and full characterization of phosphoproteins with special attention at mass spectrometry-based techniques. In particular, we will discuss affinity-based purification of phosphopeptides coupled to MALDI-TOF analysis, their detection using mass mapping and precursor ion scan, identification of modified sites by MS/MS and quantitation analysis     

Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry

Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC) coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples.     

Separation by hydrophobic interaction chromatography and structural determination by mass spectrometry of mannosylated glycoforms of a recombinant transferrin-exendin-4 fusion protein from yeast

Obtaining sufficient amounts of pure glycoprotein variants to characterize their structures is an important goal in both functional biology and the biotechnology industry. We have developed preparative HIC conditions that resolve glycoform variants on the basis of overall carbohydrate content for a recombinant transferrin-exendin-4 fusion protein. The fusion protein was expressed from the yeast Saccharomyces cerevisiae from high density fermentation and is post-translationally modified with mannose sugars through O-glycosidic linkages. Overall hydrophobic behavior appeared to be dominated by the N-terminal 39 amino acids from the exendin-4 and linker peptide sequences as compared to the less hydrophobic behavior of human transferrin alone. In addition, using LC techniques that measure total glycans released from the pure protein combined with new high resolution technologies using mass spectrometry, we have determined the locations and chain lengths of mannose residues on specific peptides derived from tryptic maps of the transferrin-exendin-4 protein. Though the protein is large (80,488 kDa) and contains 78 possible serine and threonine residues as potential sites for sugar addition, mannosylation was observed on only two tryptic peptides located within the first 55 amino acids of the N-terminus. These glycopeptides were highly heterogeneous and contained between 1 and 10 mannose residues scattered among the various serine and threonine sites which were identified by electron transfer dissociation mass spectrometry. Glycan sequences from 1 to 6 linear mannose residues were detected, but mannose chain lengths of 3 or 4 were more common and formed 80% of the total oligosaccharides. This work introduces new technological capabilities for the purification and characterization of glycosylated variants of therapeutic recombinant proteins.     

Hydrogen exchange study on the hydroxyl groups of serine and threonine residues in proteins and structure refinement using NOE restraints with polar side-chain groups

We recently developed new NMR methods for monitoring the hydrogen exchange rates of tyrosine hydroxyl (Tyr-OH) and cysteine sulfhydryl (Cys-SH) groups in proteins. These methods facilitate the identification of slowly exchanging polar side-chain protons in proteins, which serve as sources of NOE restraints for protein structure refinement. Here, we have extended the methods for monitoring the hydrogen exchange rates of the OH groups of serine (Ser) and threonine (Thr) residues in an 18.2 kDa protein, EPPIb, and thus demonstrated the usefulness of NOE restraints with slowly exchanging OH protons for refining the protein structure. The slowly exchanging Ser/Thr-OH groups were readily identified by monitoring the (13)C(β)-NMR signals in an H(2)O/D(2)O (1:1) mixture, for the protein containing Ser/Thr residues with (13)C, (2)H-double labels at their β carbons. Under these circumstances, the OH groups exist in equilibrium between the protonated and deuterated isotopomers, and the (13)C(β) peaks of the two species are resolved when their exchange rate is slower than the time scale of the isotope shift effect. In the case of EPPIb dissolved in 50 mM sodium phosphate buffer (pH 7.5) at 40 °C, one Ser and four Thr residues were found to have slowly exchanging hydroxyl groups (k(ex) < ~40 s(-1)). With the information for the slowly exchanging Ser/Thr-OH groups in hand, we could collect additional NOE restraints for EPPIb, thereby making a unique and important contribution toward defining the spatial positions of the OH protons, and thus the hydrogen-bonding acceptor atoms.     

On studying protein phosphorylation patterns using bottom-up LC-MS/MS: the case of human alpha-casein

Most proteomics studies involving mapping post-translational modifications, such as the phosphorylation of serine and threonine, are performed today using the 'bottom-up' approach. This approach involves enzymatic cleavage of proteins, most often by trypsin, with subsequent nano-LC-MS/MS. The occupancy rates of phosphosites in proteins may differ by orders of magnitude, and thus the occupancy rate must be reported for each occupied phosphosite. To highlight potential pitfalls in quantifying the occupancy rates, alpha(s1)-casein from human milk was selected as a model molecule representing moderately phosphorylated proteins. For this purpose, human milk from one Caucasian woman in the eighth month of lactation was used. The phosphorylation level of caseins is believed to have major implications for the formation of micelles that are involved in delivering valuable calcium phosphate and other minerals to the new-born. Human alpha(s1)-casein has been reported to be much less phosphorylated than ruminant caseins, which may indicate a different function of caseins in humans. Revealing the phosphorylation pattern in human casein can thus shed light on its function. The current study found that the sequence region between the residues Ser70 and Ser76 in human alpha(s1)-casein is in fact phosphorylated, contrary to previous knowledge. The site of the most abundant phosphorylation is Ser75, in agreement with the known action of the mammary gland casein kinase. There is evidence for the second phosphorylation in that region, possibly at Ser73. Earlier reported positions of phosphorylations at Ser18 and Ser26 are also confirmed, but not the dominance of Ser18 phosphorylation. The occupancy rates at Ser18, Ser26 and Ser75 are estimated to be (7 +/- 2), (20 +/- 6) and (27 +/- 9)%, respectively. Owing to differences in the ionization efficiency between phosphorylated and unphosphorylated peptides a 30% error margin is added to the occupancy rates. The highlighted pitfalls of the bottom-up strategy include the sensitivity of enzymes to proximal acidic and phosphorylated residues and the presence of multiple isoforms, including unexpected ones, of the tryptic peptides. The utility of the earlier introduced PhosTS_hunter and ModifiComb approaches for evading the latter pitfall is demonstrated.     

Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometry

A new method combining chemical modification and affinity purification is described for the characterization of serine and threonine phosphopeptides in proteins. The method is based on the conversion of phosphoserine and phosphothreonine residues to S-(2-mercaptoethyl)cysteinyl or beta-methyl-S-(2-mercaptoethyl)cysteinyl residues by beta-elimination/1,2-ethanedithiol addition, followed by reversible biotinylation of the modified proteins. After trypsin digestion, the biotinylated peptides were affinity-isolated and enriched, and subsequently subjected to structural characterization by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Database searching allowed for automated identification of modified residues that were originally phosphorylated. The applicability of the method is demonstrated by the identification of all known phosphorylation sites in a mixture of alpha-casein, beta-casein, and ovalbumin. The technique has potential for adaptations to proteome-wide analysis of protein phosphorylation.     

Identification of an in vitro insulin receptor substrate-1 phosphorylation site by negative-ion muLC/ES-API-CID-MS hybrid scan technique

Recently, we reported a fast on-line alkaline micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric approach for sensitive phosphopeptide screening of a tryptic digested protein and subsequent characterization of the identified phosphopeptide. Based on this study, we now applied an improved method for the identification of phosphorylation sites in insulin receptor substrate 1, an important mediator in insulin signal transduction which was phosphorylated in vitro by protein kinase C-zeta. The approach consists of an on-line alkaline negative-ion micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric hybrid scan experiment using a triple-quadrupole mass spectrometer with fractionation and subsequent off-line nanoES-MS (ion trap) analysis of the phosphopeptide-containing fractions. During the liquid chromatography (LC)/ES-MS experiment, the phosphopeptides of the enzymatic digest mixture of the studied insulin receptor substrate 1 fragment were detected under high skimmer potential (API-CID) using phosphorylation-specific m/z 79 marker ions as well as the intact m/z-values of the peptides which were recorded under low skimmer potential. Subsequently, the targeted fractions were analyzed by off-line nanoES-MS/MS and MS(3). Using this approach, serine 318 was clearly identified as a major in vitro protein kinase C-zeta phosphorylation site in the insulin receptor substrate -1 fragment. Together, our results indicate that the applied strategy is useful for unequivocal and fast analysis of phosphorylation sites in low abundant signaling proteins.     

Label-free relative quantification of co-eluting isobaric phosphopeptides of insulin receptor substrate-1 by HPLC-ESI-MS/MS

Intracellular signal transduction is often regulated by transient protein phosphorylation in response to external stimuli. Insulin signaling is dependent on specific protein phosphorylation events, and analysis of insulin receptor substrate-1 (IRS-1) phosphorylation reveals a complex interplay between tyrosine, serine, and threonine phosphorylation. The phosphospecific antibody-based quantification approach for analyzing changes in site-specific phosphorylation of IRS-1 is difficult due to the dearth of phospho-antibodies compared with the large number of known IRS-1 phosphorylation sites. We previously published a method detailing a peak area-based mass spectrometry approach, using precursor ions for peptides, to quantify the relative abundance of site-specific phosphorylation in the absence or presence of insulin. We now present an improvement wherein site-specific phosphorylation is quantified by determining the peak area of fragment ions respective to the phospho-site of interest. This provides the advantage of being able to quantify co-eluting isobaric phosphopeptides (differentially phosphorylated versions of the same peptide), allowing for a more comprehensive analysis of protein phosphorylation. Quantifying human IRS-1 phosphorylation sites at Ser303, Ser323, Ser330, Ser348, Ser527, and Ser531 shows that this method is linear (n = 3; r² = 0.85 ± 0.05, 0.96 ± 0.01, 0.96 ± 0.02, 0.86 ± 0.07, 0.90 ± 0.03, 0.91 ± 0.04, respectively) over an approximate 10-fold range of concentrations and reproducible (n = 4; coefficient of variation = 0.12, 0.14, 0.29, 0.30, 0.12, 0.06, respectively). This application of label-free, fragment ion-based quantification to assess relative phosphorylation changes of specific proteins will prove useful for understanding how various cell stimuli regulate protein function by phosphorylation.     

Quantification of phosphorylation of insulin receptor substrate-1 by HPLC-ESI-MS/MS

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means. A mass spectrometry peak area based quantification approach has been developed and applied to assess the relative abundance of IRS-1 phosphorylation in the absence or presence of stimuli. In this method, the peak area for a phosphopeptide of interest is normalized against the average of peak areas for six selected representative IRS-1 peptides that serve as endogenous internal standards. Relative quantification of each phosphopeptide is then obtained by comparing the normalized peak area ratios for untreated and treated samples. Two non-IRS-1 peptides were added to each digest for use as HPLC retention time markers and additional standards as well as references to the relative quantity of IRS-1 in different samples. This approach does not require isotopic or chemical labeling and can be applied to various cell lines and tissues. Using this method, we assessed the relative changes in the quantities of two tryptic phosphopeptides isolated from human IRS-1 expressed in L6 cells incubated in the absence or presence of insulin or tumor necrosis factor-alpha. Substantial increases of phosphorylation were observed for Thr(446) upon stimulation. In contrast, no obvious change in the level of phosphorylation was observed for Ser(1078). This mass spectrometry based strategy provides a powerful means to quantify changes in the relative phosphorylation of peptides in response to various stimuli in a complex, low-abundance protein.     

On-line capillary liquid chromatography tandem mass spectrometry on an ion trap/ reflectron time-of-flight mass spectrometer using the sequence tag database search approach for peptide sequencing and protein identification

Capillary high-performance liquid chromatography has been coupled on-line with an ion trap storage/reflectron time-of-flight mass spectrometer to perform tandem mass spectrometry for tryptic peptides. Selection and fragmentation of the precursor ions were performed in a three-dimensional ion trap, and the resulting fragment ions were pulsed out of the trap into a reflectron time-of-flight mass spectrometer for mass analysis. The stored waveform inverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collision-induced dissociation. Tandem mass spectra of various doubly charged tryptic peptides were investigated where a conspicuous y ion series over a certain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by database search using the sequence tag approach. Several peptides from tryptic digests of horse heart myoglobin and bovine cytochrome c were selected for tandem mass spectrometry (MS/MS) where it was demonstrated that the proteins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimensional gel separation of a human esophageal adenocarcinoma cell line.     

A database of 660 peptide ion cross sections: Use of intrinsic size parameters for bona fide predictions of cross sections

An ion trap/ion mobility/time-of-flight mass spectrometry technique has been used to measure collision cross sections for 660 peptide ions generated by tryptic digestion of 34 common proteins. Measured cross sections have been compiled into a database that contains peptide molecular weight and sequence information. The database is used to generate average intrinsic contributions to cross section (size parameters) for different amino acid residues by solving systems of equations that relate the unknown contributions of individual residues to the sequences and cross sections of database peptides. Size parameters are combined with information about amino acid composition to calculate cross sections for database peptides. Bona fide cross section predictions (made prior to measurement) for peptides observed in tryptic digests of sperm whale myoglobin and yeast enolase are made. Eight of 10 predicted cross sections are within 2% of the experimental values and all 10 are within 3.2%. The utility of size parameters for cross section prediction is explored and discussed.     

Chemical derivatization of peptides containing phosphorylated serine/threonine for efficient ionization and quantification in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We describe a useful method for the efficient ionization and relative quantification of peptides containing serine/threonine phosphorylation sites. This method is based on beta-elimination of the phosphate group from serine/threonine phosphorylation sites under alkaline conditions, followed by Michael addition reaction with N-(2-mercaptoethyl)-6-methylnicotinamide (MEMN). As a result of the derivatization reaction, the negatively charged phosphate group is substituted with the nicotinoyl moiety to improve the ionization efficiency of the derivatized peptide. The combination of d(3)-labeled MEMN (d(3)-MEMN) and MEMN (d(0)-MEMN) generates a 3 Da mass difference between d(3)-MEMN-labeled and d(0)-MEMN-labeled peptides, which is a useful signature for the identification of peptides containing serine/threonine phosphorylation sites in the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum. Moreover, the mass difference is useful for the quantitative analysis of serine/threonine phosphorylation in proteins. In this paper, we describe the synthesis of d(0)/d(3)-labeled MEMN and an application of our approach to model peptides and proteins.     

A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E. coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron resonance mass spectrometer

A sensitive, integrated top-down liquid chromatography/mass spectrometry (LC/MS) approach, suitable for the near complete characterization of specific proteins in complex protein mixtures, such as inclusion bodies of an E. coli lysate, has been successfully developed using a hybrid linear ion trap/Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. In particular, human growth hormone (hGH) (200 fmol) was analyzed with high sequence coverage (>95%), including the sites of disulfide linkages. The high mass accuracy and resolution of the FTICR mass spectrometer was used to reveal high charge state ions of hGH (22 kDa). The highly charged intact protein ions (such as the 17+ species) were captured and fragmented in the linear ion trap cell. The fragment ions from MS/MS spectra were then successfully analyzed in the FTICR cell in an on-line LC/MS run. Peptide fragments from the N-terminal and C-terminal regions, as well as large interior fragments, were captured and identified. The results allowed the unambiguous assignment of disulfide bonds Cys53-Cys165 and Cys182-Cys189, indicative of proper folding of hGH. The disulfide bond assignments were also confirmed by analysis of the tryptic digest of a sample of hGH purified from inclusion bodies. On-line LC/MS with the linear ion trap/FTICR yields high mass accuracy in both the MS and MS/MS modes (within 2 ppm with external calibration). The approach should prove useful in biotechnology applications to characterize correctly folded proteins, both in the early protein expression and the later processed stages, using only a single automated on-line LC/MS top-down method.     

An efficient approach for the characterization of mucin-type glycopeptides: the effect of O-glycosylation on the conformation of synthetic mucin peptides

Despite the growing importance of mucin core O-glycosylation in many biological processes including the protection of epithelial cell surfaces, the immune response, cell adhesion, inflammation, and tumorigenesis/metastasis, the regulation mechanism and conformational significance of the multiple introduction of α-GalNAc residues by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) remains unclear. Here we report an efficient approach by combining MS and NMR spectroscopy that allows for the identification of O-glycosylation site(s) and the effect of O-glycosylation on the peptide backbone structures during enzymatic mucin domain assembly by using an isoform UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T2 (ppGalNAcT2) in vitro. An electron-capture dissociation device in a linear radio-frequency quadrupole ion trap (RFQ-ECD) combined with a time-of-flight (TOF) mass spectrometer was employed for the identification of Thr/Ser residues occupied by α-GalNAc branching among multiple and potential O-glycosylation sites in the tandem repeats of human mucin glycoproteins MUC4 (Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp) and MUC5AC (Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly). In the present study, O-glycosylation was initiated specifically at Thr10 in naked MUC4 peptide and additional introduction of α-GalNAc proceeded preferentially but randomly at three other Thr residues to afford densely glycosylated MUC4 containing six α-GalNAc residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15. On the contrary, O-glycosylation of naked MUC5AC peptide occurred predominantly at consecutive Thr residues and led to MUC5AC with four α-GalNAc residues at Thr2, Thr3, Thr7, and Thr8. The solution structures determined by NMR spectroscopic studies elicited that the preferential introduction of α-GalNAc at Thr10 of MUC4 stabilizes specifically a β-like extended backbone structure at this area, whereas other synthetic models with a single α-GalNAc residue at Thr1, Thr6, or Thr15 did not exhibit any converged three-dimensional structure at the proximal peptide moiety. Such conformational impact on the underlying peptides was proved to be remarkable in the glycosylation at the consecutive Thr residues of MUC5AC.     

Beam-type collisional activation of polypeptide cations that survive ion/ion electron transfer

Doubly protonated peptides that undergo an electron transfer reaction without dissociation in a linear ion trap can be subjected to beam-type collisional activation upon transfer from the linear ion trap into an adjacent mass analyzer, as demonstrated here with a hybrid triple quadrupole/linear ion trap system. The activation can be promoted by use of a DC offset difference between the ion trap used for reaction and the ion trap into which the products are injected of 12-16 V, which gives rise to energetic collisions between the transferred ions and the collision/bath gas employed in the linear ion trap used for ion/ion reactions. Such a process can be executed routinely on hybrid linear ion trap/triple quadrupole tandem mass spectrometers and is demonstrated here with several model peptides as well as a few dozen tryptic peptides. Collisional activation of the peptide precursor ions that survive electron transfer frequently provides structural information that is absent from the precursor ions that fragment spontaneously upon electron transfer. The degree to which additional structural information is obtained by collisional activation of the surviving singly charged peptide ions depends upon peptide size. Little or no additional structural information is obtained from small peptides (<8 residues) due to the high electron transfer dissociation (ETD) efficiencies noted for these peptides as well as the extensive sequence information that tends to be forthcoming from ETD of such species. Collisional activation of the surviving electron transfer products provided greatest benefit for peptides of 8-15 residues.     

Increased expression and phosphorylation of liver glutamine synthetase in well-differentiated hepatocellular carcinoma tissues from patients infected with hepatitis C virus

Hepatocellular carcinoma (HCC) is one of the most common fatal cancers, and chronic infection with hepatitis C virus (HCV) is thought to be one of the main causes in Japan. To identify diagnostic or therapeutic biomarkers for HCC associated with HCV (HCV-HCC), we tried to elucidate the factors related to the products from cancerous tissues of HCV-infected patients. From proteomic differential display analysis of liver tissue samples from HCV-HCC cancerous tissues and corresponding non-cancerous tissues from patients, three protein spots of the same molecular mass (42 kDa), whose expression increased in well-differentiated cancerous tissues, were detected. Although their pI were different, they were identified as glutamine synthetase (GS) by PMF with MALDI-TOF MS and by Western blotting using anti-GS specific mAb. Immunohistochemical analysis showed that tumor tissue consists of two parts, GS-positive cell and GS-negative cell regions, suggesting that GS-producing cells grew in the tumor tissue as a nodule in nodules. The tryptic peptides of the most acidic GS isoform lost the signal of 899.5 Da, corresponding a peptide of SASIRIPR, and gained a signal of 1059.5 Da, which was submitted to PSD analysis. PSD analysis showed the neutral loss by elimination of two phosphate groups, supposed to be on serine residues of the 899.5-Da peptide, from serine 320 to arginine 327 in GS. PMF followed by PSD analysis is thought to be useful for the determination of phosphorylation sites of proteins showing molecular heterogeneity.     

Analysis on heat stress-induced hyperphosphorylation of stathmin at serine 37 in Jurkat cells by means of two-dimensional gel electrophoresis and tandem mass spectrometry

Two-dimensional gel electrophoresis (2-DE) and tandem mass spectrometry were successfully used for determination of a phosphorylation site of stathmin induced by heat stress to Jurkat cells of a human T lymphoblastic cell line. The cells were incubated for 30 min at 41 degrees C up to 45 degrees C in a serum free 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered culture medium. The intracellular soluble proteins were separated by 2-DE, and some of the proteins increased their abundance by heat stress. Those proteins were identified to be calmodulin, protein kinase C substrate, thymosin beta-4 and F-actin capping protein beta-subunit by peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). On the contrary, protein phosphatase 2C gamma-isoform, nucleophosmin, translationally controlled tumor protein, Rho GDP-dissociation inhibitor-1, eukaryotic translation initiation factors 5A and 3A subunit 2, ubiquitin-like protein SMT 3B and chloride intracellular channel protein-1 were decreased their abundance. A protein spot of M(r) 18,000 and pI 5.9 was markedly increased at temperatures higher than 43 degrees C at which the cells were led to apoptosis. The spot was identified to be stathmin of a signal relay protein which has a function of sequestering microtubule. MALDI-quadrupole ion trap (QIT)-TOF-MS/MS and immunoblotting with a monoclonal antibody specific for a phosphorylation site of stathmin showed that the spot was a phosphorylated stathmin at serine 37 (Ser 37). The phosphorylation was suppressed by treatment of cells with olomoucine of an inhibitor specific for cyclin dependent kinase (Cdk-1). These results strongly suggest that heat stress activates Cdk-1 which phosphorylates Ser 37 on the stathmin molecule. The phosphorylation may cause the functional loss of stathmin for dynamic microtubule assembly and leads Jurkat cells to cell cycle arrest and apoptosis.