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.     

Analysis of protein tyrosine phosphorylation by nanoelectrospray ionization high-resolution tandem mass spectrometry and tyrosine-targeted product ion scanning

A novel highly sensitive strategy is introduced for analysis of tyrosine phosphorylation in previously identified proteins channelling for this aim all analytical and sequence information available. Nanoelectrospray high-resolution MS/MS analysis is targeted to precalculated m/z values corresponding to phosphotyrosine-containing tryptic peptides. Identification of these peptides is supported by the occurrence of the phosphotyrosine immonium ion at m/z 216, neutral loss of 79.97/z (= loss of HPO3), and similarity of the fragmentation patterns of phosphotyrosine-containing peptides with their nonphosphorylated analogues. This tyrosine-targeted tandem mass spectrometry strategy is demonstrated for epidermal growth factor receptor showing that phosphotyrosine-containing tryptic peptides invisible in the survey spectrum can be safely identified.     

Screening and identification of glycosides in biological samples using energy-gradient neutral loss scan and liquid chromatography tandem mass spectrometry

A rapid, selective, and reliable strategy has been developed for the screening and identification of glycosides in biological samples: a crude extract was directly infused to a triple-quadrupole MS/MS, and major glycosides were screened out with high confidence by an energy-gradient neutral loss scan (EGNLS) for the loss of sugar(s); then these glycosides were further identified with LC/MS/MS. The proposed EGNLS method was established and optimized with 16 representative glycosides (including ginsenosides and the glycosides of flavones, anthraquinones, and terpenoids). The EGNLS method has two major advantages over the conventional fixed-energy neutral loss scan: (1) The latter is liable to '"omit" some target compounds due to the usual mismatch between the preset collision energy and interested compounds' optimal collision energy (OCE), while EGNLS solves this problem by scanning over an energy range. (2) The EGNLS simultaneously measures the screened compounds' OCE, which not only are essential parameters for further LC/MS/MS analysis but also carry some structural information, as proved by this study. This strategy has been successfully demonstrated with the analysis of glycosides in Scutellaria viscidula Bge and transformed Panaxhairy roots (the glycoside constitutions of both had not been studied before): without laborious separation processes; comprehensive glycoside information on those two plants was obtained by a rapid and simple procedure. This strategy is valuable for the study of glycosides in complex samples.     

Analysis of derivatized biogenic aldehydes by LC tandem mass spectrometry

Lipid peroxidation has been linked to the etiology of several diseases, including Alzheimer's disease (AD). End products of this phenomenon include low molecular weight, water-soluble aldehydes, compounds that covalently modify proteins and nucleic acids, thereby altering function. Aliphatic aldehydes (C3-C10) are generated during lipid peroxidation, along with alpha,beta-unsaturated aldehydes, including acrolein and 4-hydroxynonenal (HNE). The Hantzsch reaction was used to produce heterocyclic aldehyde derivatives that can be conveniently analyzed with mass spectrometry. Liquid chromatographic analyses revealed increasing retention times from derivatized methanal to octanal. HNE derivatives were observed to elute between heptanal and octanal derivatives, while the acrolein derivatives had a retention time similar to the propanal derivative. Smaller aliphatic aldehyde derivatives fragmented in a similar manner to produce a base peak of m/z 273, while the larger derivatives yielded m/z 274 as the base peak. Acrolein and HNE derivatives fragmented in a slightly different manner compared to their aliphatic counterparts. Calibration plots of aliphatic and unsaturated aldehydes were linear (r2 >/= 0.99) in the concentration range explored (approximately 5-1500 pg on column). The LC-MS/MS methodology developed here will be used in subsequent studies to determine aldehyde concentrations for comparing age-matched controls to AD tissues from human subjects.     

Analysis of nonderivatized neutral and sialylated oligosaccharides by electrospray mass spectrometry

An HPLC/MS method has been developed that allows rapid, direct analysis of underivatized sialylated as well as neutral oligosaccharides. The method involves the separation of oligosaccharides from salts and proteins using RP-HPLC with a formic acid/acetonitrile/water mobile phase system and on-line electrospray mass spectrometry analysis in the positive ion mode. Under the solution conditions employed, both neutral and acidic (sialylated) oligosaccharides are protonated and therefore detected. In contrast to MALDI-TOF MS, no loss of sialic acid is observed when operating in the positive ion mode. Furthermore, the capability of this method to provide quantitative estimates of the relative abundance of each oligosaccharide mass has been demonstrated using fetuin as a model compound.     

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.     

Structure elucidation of DNA interstrand cross-link by a combination of nuclease P1 digestion with mass spectrometry

DNA interstrand cross-link reagents are among the most powerful agents for cancer treatment. Here we report a combined nuclease P1 digestion/mass spectrometry method for the structure elucidation of duplex oligodeoxynucleotides (ODNs) containing an interstrand cross-link. Our results demonstrate that nuclease P1 digestion of a double-stranded ODN containing an interstrand cross-link (ICL) of 4,5',8-trimethylpsoralen or mitomycin C gives a tetranucleotide bearing the cross-linked nucleobase moiety. Product ion spectra of the deprotonated ions of the tetranucleotides provide information about the structure of the cross-link. Furthermore, product-ion spectra of tetranucleotides containing two orientation isomers of mitomycin C interstrand cross-link are distinctive. We believe that the method described in this paper can be generally applicable for investigating the structures of other DNA ICLs.     

Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked beta-(1-4) and beta-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS. In MS(1) spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS(2) and MS(3) spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolation of individual oligosaccharides by HPLC or other methods.     

STAT: a saccharide topology analysis tool used in combination with tandem mass spectrometry

Sequential stages of mass spectrometry (MSn) have the potential to provide a great deal of structural information in glycan analysis. The saccharide topology analysis tool (STAT) presented here is a Web-based computational program that can quickly extract sequence information from a set of MSn spectra for an oligosaccharide of up to 10 residues. After information such as precursor ion mass, possible monosaccharide moieties, charge carrier, and product ion mass has been input, all possible connectivities are generated and evaluated against the MSn data. The list of possible structures is given a rating based on the likelihood that it is the correct sequence. Examples are given to demonstrate the feasibility of applying STAT to MSn data generated from bacterial lipooligosaccharides and an N-linked glycan. The major advantage of STAT is that the list of possible structures is generated quickly and the rating system pushes the more likely structures to the top of the list. Combining the data generated by STAT with data on the branching patterns of the glycan serves to eliminate all but a handful of structures. These remaining structures could then be used to guide further structural analysis.     

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.     

An on-line protein digestion device for rapid peptide mapping by electrospray mass spectrometry

A simple laboratory-constructed device has been developed for fast on-line protein digestion followed by peptide mapping by use of electrospray mass spectrometry. Taking advantage of its nonsolubility properties at near-neutral pH values, pepsin could be nonpermanently attached to the PEEK tube commonly employed as transfer capillary between the syringe and the electrospray ion source. After optimization of experimental conditions such as pH, solvent, and exposure time, efficient digestion of several model proteins of molecular weights between 14,000 and 66,000, some having disulfide bridges, was successfully carried out. This technique provided reliable and reproducible sequence information by means of C-terminal-specific cleavages of aromatic and hydrophobic residues. As an application, protein identification could be achieved using a protein database search software.     

Quantitative analysis of protein phosphorylation in mouse brain by hypothesis-driven multistage mass spectrometry

Determination of site-specific changes in the levels of protein phosphorylation in mammals presents a formidable analytical challenge. Here, we demonstrate a strategy for such analyses utilizing a combination of stable isotope chemical labeling and tandem mass spectrometry. Phosphoproteins of interest are isolated from two sets of animals that have undergone differential drug treatments, separated by SDS-PAGE, excised, and subjected to in-gel enzymatic digestion. Using a simple chemical labeling step, we introduce stable, isotopically distinct mass tags into each of the two sets of peptides that originate from the samples under comparison, mix the samples, and subject the resulting mixture to a procedure based on our previously reported hypothesis-driven multistage MS (HMS-MS) method (Chang, E. J.; Archambault, V.; McLachlin, D. T.; Krutchinsky, A. N.; Chait, B. T. Anal. Chem. 2004, 76, 4472-4483). The method takes advantage of the dominant loss of H3PO4 during MS/MS from singly charged phosphopeptide ions produced by matrix-assisted laser desorption/ionization (MALDI) in the ion trap mass spectrometer. In the present work, quantitation is achieved by isolating the range of m/z values that include both isotopic forms of the putative phosphopeptide and measuring the relative intensities of the two resulting -98-Da fragment ion peaks. This MS/MS measurement can be repeated on the same MALDI sample for all potential phosphopeptide ion pairs that we hypothesize might be produced from the protein under study. Use of MS/MS for quantitation greatly increases the sensitivity of the method and allows us to measure relatively low levels of phosphorylation, phosphopeptides, or both that are not easily observable by single-stage MS. We apply the current method to the determination of changes in the levels of phosphorylation in DARPP-32 from the mouse striatum upon treatment of animals with psychostimulant drugs.     

Mass spectrometry and tandem mass spectrometry of citrus limonoids

Methods for atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) of citrus limonoid aglycones and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of limonoid glucosides are reported. The fragmentation patterns of four citrus limonoid aglycones (limonin, nomilin, obacunone, and deacetylnomilin) and six limonoid glucosides, that is, limonin 17-beta-D-glucopyranoside (LG), nomilin 17-beta-D-glucopyranoside (NG), nomilinic acid 17-beta-D-glucopyranoside (NAG), deacetyl nomilinic acid 17-beta-D-glucopyranoside (DNAG), obacunone 17-beta-D-glucopyranoside (OG), and obacunoic acid 17-beta-D-glucopyranoside (OAG) were investigated using a quadruple mass spectrometer in low-energy collisionally activated dissociation (CAD). The four limonoid aglycones and four limonoid glucosides (LG, OG, NAG, and DNAG) were purified from citrus seeds; the other two limonoid glucosides (NG and OAG) were tentatively identified in the crude extract of grapefruit seeds by ESI mass spectrometry in both positive and negative ion analysis. Ammonium hydroxide or acetic acid was added to the mobile phase to facilitate ionization. During positive ion APCI analysis of limonoid aglycones, protonated molecular ion, [M + H]+, or adduct ion, [M + NH3 + H]-, was formed as base peaks when ammonium hydroxide was added to the mobile phase. Molecular anions or adduct ions with acetic acid ([M + HOAc - H] and [M + HOAc]-) or a deprotonated molecular ion were produced during negative ion APCI analysis of limonoid aglycones, depending on the mobile-phase modifier used. Positive ion ESI-MS of limonoid glucosides produced adduct ions of [M + H + NH3]+, [M + Na]+, and [M + K]+ when ammonium hydroxide was added to the mobile phase. After collisionally activated dissociation (CAD) of the limonoid aglycone molecular ions in negative ion APCI analysis, fragment ions indicated structural information of the precursor ions, showing the presence of methyl, carboxyl, and oxygenated ring structure. CAD of the adduct ion [M + H + NH3]+ of limonoid glucosides produced the aglycone moiety corresponding to each glucoside. The combination of mass spectrometry and tandem mass spectrometry provides a powerful technique for identification and characterization of citrus limonoids.     

Characterization of hydrophobic peptides by atmospheric pressure photoionization-mass spectrometry and tandem mass spectrometry

The use of photoionization at atmospheric pressure shows great potential for the mass analysis of large apolar or hydrophobic peptides. Mass spectra that were obtained using this technique showed mainly singly charged ions. While polar peptides spectra do not produce fragment ions, others lead to B-type or C-type in-source fragmentation. These dissociation reactions, which could involve electron capture dissociation processes in the case of the C-type ions, are observed for hydrophobic peptides. Both the compatibility of this ionization mode with reversed- or normal-phase liquid chromatographic separation and its sensitivity allow liquid chromatography coupling to both mass spectrometry and tandem mass spectrometry for the analyses of hydrophobic peptide mixtures. Atmospheric pressure photoionization seems to be an interesting alternative method to study hydrophobic peptides that are not easily ionizable by more classical ionization techniques such as electrospray ionization and matrix-assisted laser desorption/ionization.     

Spatially resolved protein hydrogen exchange measured by subzero-cooled chip-based nanoelectrospray ionization tandem mass spectrometry

Mass spectrometry has become a valuable method for studying structural dynamics of proteins in solution by measuring their backbone amide hydrogen/deuterium exchange (HDX) kinetics. In a typical exchange experiment one or more proteins are incubated in deuterated buffer at physiological conditions. After a given period of deuteration, the exchange reaction is quenched by acidification (pH 2.5) and cooling (0 °C) and the deuterated protein (or a digest thereof) is analyzed by mass spectrometry. The unavoidable loss of deuterium (back-exchange) that occurs under quench conditions is undesired as it leads to loss of information. Here we describe the successful application of a chip-based nanoelectrospray ionization mass spectrometry top-down fragmentation approach based on cooling to subzero temperature (-15 °C) which reduces the back-exchange at quench conditions to very low levels. For example, only 4% and 6% deuterium loss for fully deuterated ubiquitin and β(2)-microglobulin were observed after 10 min of back-exchange. The practical value of our subzero-cooled setup for top-down fragmentation HDX analyses is demonstrated by electron-transfer dissociation of ubiquitin ions under carefully optimized mass spectrometric conditions where gas-phase hydrogen scrambling is negligible. Our results show that the known dynamic behavior of ubiquitin in solution is accurately reflected in the deuterium contents of the fragment ions.     

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.     

A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry

Recent advances in phosphopeptide enrichment prior to mass spectrometric analysis show genuine promise for characterization of phosphoproteomes. Tandem mass spectrometry of phosphopeptide ions, using collision-activated dissociation (CAD), often produces product ions dominated by the neutral loss of phosphoric acid. Here we describe a novel method, termed Pseudo MS(n), for phosphopeptide ion dissociation in quadrupole ion trap mass spectrometers. The method induces collisional activation of product ions, those resulting from neutral loss(es) of phosphoric acid, following activation of the precursor ion. Thus, the principal neutral loss product ions are converted into a variety of structurally informative species. Since product ions from both the original precursor activation and all subsequent neutral loss product activations are simultaneously stored, the method generates a "composite" spectrum containing fragments derived from multiple precursors. In comparison to analysis by conventional MS/MS (CAD), Pseudo MS(n) shows improved phosphopeptide ion dissociation for 7 out of 10 synthetic phosphopeptides, as judged by an automated search algorithm (TurboSEQUEST). A similar overall improvement was observed upon application of Pseudo MS(n) to peptides generated by enzymatic digestion of a single phosphoprotein. Finally, when applied to a complex phosphopeptide mixture, several phosphopeptides mis-assigned by TurboSEQUEST under the conventional CAD approach were successfully identified after analysis by Pseudo MS(n).     

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.     

Lookup peaks: a hybrid of de novo sequencing and database search for protein identification by tandem mass spectrometry

A powerful technique for peptide and protein identification is tandem mass spectrometry followed by database search using a program such as SEQUEST or Mascot. These programs, however, become slow and lose sensitivity when allowing nonspecific cleavages or peptide modifications. De novo sequencing and hybrid methods such as sequence tagging offer speed and robustness for wider searches, yet these approaches require better spectra with more complete and consecutive fragmentation and, hence, are less sensitive to low-abundance peptides. Here we describe a new hybrid method that retains the sensitivity of pure database search. The method uses a small amount of de novo analysis to identify likely b- and y-ion peaks--"lookup peaks"--that can then be used to extract candidate peptides from the database, with the number of candidates tunable to fit a computing budget. We describe a program called ByOnic that implements this method, and we benchmark ByOnic on several data sets, including one of mouse blood plasma spiked with low concentrations of recombinant human proteins. We demonstrate that ByOnic is more sensitive than sequence tagging and, indeed, more sensitive than the three most popular pure database search tools--SEQUEST, Mascot, and X!Tandem--on both the peptide and protein levels. On the mouse plasma samples, ByOnic consistently found spiked proteins missed by the other tools.     

Rapid screening for taxanes by tandem mass spectrometry.

A highly specific and sensitive method is described for determining taxol, cephalomannine, and baccatin III in crude plant extracts. Radical anions of the taxanes are formed by desorption chemical ionization, and a parent tandem mass spectrometric scan is used to recognize these compounds by their characteristic dissociations. The limit of detection of the individual taxanes in typical plant matrices is less than 500 pg when all three species are screened simultaneously. Because of the sensitivity of the method, extraction times can be shortened to 30 min and crude extracts can be examined at the rate of 6/h. Detection of all three taxanes extracted from a single Taxus cuspidata needle in a combined extraction/analysis time of less than 1 h is demonstrated.