Acid-labile isotope-coded extractants: a class of reagents for quantitative mass spectrometric analysis of complex protein mixtures

Quantitative mass spectrometry using stable isotope-labeled tagging reagents such as isotope-coded affinity tags has emerged as a powerful tool for identification and relative quantitation of proteins in current proteomic studies. Here we describe an integrated approach using both automated two-dimensional liquid chromatography/ mass spectrometry (2D-LC/MS) and a novel class of chemically modified resins, termed acid-labile isotope-coded extractants (ALICE), for quantitative mass spectrometric analysis of protein mixtures. ALICE contains a thiol-reactive group that is used to capture all cysteine (Cys)-containing peptides from peptide mixtures, an acid-labile linker, and a nonbiological polymer. The acid-labile linker is synthesized in both heavy and light isotope-coded forms and therefore enables the direct relative quantitation of peptides/proteins through mass spectrometric analysis. To test the ALICE method for quantitative protein analysis, two model protein mixtures were fully reduced, alkylated, and digested in solution separately and then Cys-containing peptides covalently captured by either light or heavy ALICE. The reacted light and heavy ALICE were mixed and washed extensively under rigorous conditions and the Cys-containing peptides retrieved by mild acid-catalyzed elution. Finally, the eluted peptides were directly subjected to automated 2D-LC/MS for protein identification and LC/MS for accurate relative quantitation. Our initial study showed that quantitation of protein mixtures using ALICE was accurate. In addition, isolation of Cys-containing peptides by the ALICE method was robust and specific and thus yielded very low background in mass spectrometric studies. Overall, the use of ALICE provides improved dynamic range and sensitivity for quantitative mass spectrometric analysis of peptide or protein mixtures.     

Combining fluorescence detection and mass spectrometric analysis for comprehensive and quantitative analysis of redox-sensitive cysteines in native membrane proteins

Monobromobimane (MBB) is a lipophilic reagent that selectively modifies free cysteine residues in proteins. Because of its lipophilic character, MBB is capable of labeling cysteine residues in membrane proteins under native conditions. Reaction of MBB with the sulfhydryl groups of free cysteines leads to formation of highly fluorescent derivatives. Here we describe a procedure for the detection and relative quantitation of MBB-labeled cysteines using fluorescence and mass spectrometric analyses, which allow determination of free cysteine content and unambiguous identification of MBB-modified cysteine residues. We have applied this approach to the analysis of the free and redox-sensitive cysteine residues of a large membrane protein, the sarcoplasmic reticulum Ca2+ release channel with a molecular mass of 2.2 million Da. Labeling was performed under physiologic conditions where the channel complex is in its native environment and is functionally active. The purified MBB-labeled channel complex was enzymatically digested, and the resulting peptides were separated by reversed-phase high-performance chromatography. MBB-labeled peptides were detected by fluorescence and identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Under MALDI conditions, partial photolytic fragmentation of the MBB-peptide bound occurred, thus allowing convenient screening for the MBB-modified peptides in the MS spectrum by detection of the specific mass increment of 190.07 Da for MBB-modified cysteine residues. Modification of the peptides was further confirmed by tandem mass spectrometric analysis, utilizing sequencing information and the presence of the specific immonium ion for the MBB-modified cysteine residues at m/z 266.6. Quantitative information was obtained by comparison of both fluorescence and MS signal intensities of MBB-modified peptides. Combination of fluorescence with MS detection and analysis of MBB-labeled peptides supported by a customized software program provides a convenient method for identifying and quantifying redox-sensitive cysteines in membrane proteins of native biological systems. Identification of one redox-sensitive cysteine (2327) in the native membrane-bound sarcoplasmic reticulum Ca2+ release channel is described.     

Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards

Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standards. Synthetic peptides are designed to mimic peptides that are cleaved from target analyte proteins by proteolytic or chemical digestion, and the peptides selected serve as standards for quantification by LC/MS/MS on a triple quadrupole mass spectrometer. The technique is complementary to relative quantification techniques in widespread use by providing absolute quantitation of selected targets with greater sensitivity, dynamic range, and precision. Proteins that are found to be of interest by global proteome searches can be selected as targets for quantitation by the present method. This method has a much shorter analytical cycle time (minutes versus hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach using synthetic peptides as standards, in conjunction with proteolytic or chemical cleavage of target proteins, allows mass spectrometry to be used as a highly selective detector for providing absolute quantification of proteins for which no standards are available. We demonstrate that quantification is simple and reliable for the integral membrane protein rhodopsin with reasonable recoveries for replicate experiments using low-micromolar solutions of rhodopsin from rod outer segments.     

Oxygen isotopic substitution of peptidyl phosphates for modification-specific mass spectrometry

The first method of isotopic substitution of a nonbridging oxygen atom in pre-existing phosphates on peptides is reported, solving a long-standing, challenging issue in the sample preparation of phosphopeptides. Peptidyl phosphates, phosphate groups on phosphopeptides, are converted to phosphoramidates with carbodiimide assistance. Acid-catalyzed hydrolysis of the newly formed phosphoramidates incorporates one oxygen atom from H2(16)O or H2(18)O, producing peptidyl phosphates-16O1 or -18O1, respectively. The oxygen labels are stable under common separation and analysis conditions. This labeling method causes minimal structural alteration to peptidyl phosphates and allows the direct application of established phosphate-specific marker ions to the mass spectrometric analysis of differentially labeled phosphopeptide pairs. Using phosphotyrosinyl peptides as model analytes, the characteristic 16O1- and 18O1-labeled phosphotyrosine immonium ions at m/z 216.043 and 218.047 are used for developing a method of phosphopeptide quantitation that is independent of the amino acid sequence of the peptides. From analysis by tandem parallel fragmentation mass spectrometry, it is clear that the phosphate-specific marker ions authentically inherit the quantitative information from precursor phosphopeptides. The dynamic range for relative quantitation of differentially labeled phosphopeptides is at least 2 orders of magnitude for experiments run on a quadrupole time-of-flight mass spectrometer. The use of 16O1 and 18O1 labeling for counting the number of phosphate groups on peptides is also demonstrated.     

Mass defect labeling of cysteine for improving peptide assignment in shotgun proteomic analyses

A method for improving the identification of peptides in a shotgun proteome analysis using accurate mass measurement has been developed. The improvement is based upon the derivatization of cysteine residues with a novel reagent, 2,4-dibromo-(2'-iodo)acetanilide. The derivitization changes the mass defect of cysteine-containing proteolytic peptides in a manner that increases their identification specificity. Peptide masses were measured using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron mass spectrometry. Reactions with protein standards show that the derivatization of cysteine is rapid and quantitative, and the data suggest that the derivatized peptides are more easily ionized or detected than unlabeled cysteine-containing peptides. The reagent was tested on a 15N-metabolically labeled proteome from M. maripaludis. Proteins were identified by their accurate mass values and from their nitrogen stoichiometry. A total of 47% of the labeled peptides are identified versus 27% for the unlabeled peptides. This procedure permits the identification of proteins from the M. maripaludis proteome that are not usually observed by the standard protocol and shows that better protein coverage is obtained with this methodology.     

Cleavage of peptides and proteins using light-generated radicals from titanium dioxide

Protein identification and characterization often requires cleavage into distinct fragments. Current methods require proteolytic enzymes or chemical agents and typically a second reagent to discontinue cleavage. We have developed a selective cleavage process for peptides and proteins using light-generated radicals from titanium dioxide. The hydroxyl radicals, produced at the TiO(2) surface using UV light, are present for only hundreds of microseconds and are confined to a defined reagent zone. Peptides and proteins can be moved past the "reagent zone", and cleavage is tunable through residence time, illumination time, and intensity. Using this method, products are observed consistent with cleavage at proline residues. These initial experiments indicate the method is rapid, specific, and reproducible. In certain configurations, cleavage products are produced in less than 10 s. Reproducible product patterns consistent with cleavage of the peptide bond at proline for angiotensin I, Lys-bradykinin, and myoglobin are demonstrated using capillary electrophoresis. Mass characterization of fragments produced in the cleavage of angiotensin I was obtained using liquid chromatography-mass spectrometry. In addition to the evidence supporting cleavage at proline, enkephalin and peptide A-779, two peptides that do not contain proline, showed no evidence of cleavage under the same conditions.     

Method for quantitative proteomics research by using metal element chelated tags coupled with mass spectrometry

The mass spectrometry-based methods with a stable isotope as the internal standard in quantitative proteomics have been developed quickly in recent years. But the use of some stable isotope reagents is limited by the relative high price and synthetic difficulties. We have developed a new method for quantitative proteomics research by using metal element chelated tags (MECT) coupled with mass spectrometry. The bicyclic anhydride diethylenetriamine-N,N,N',N' ',N' '-pentaacetic acid (DTPA) is covalently coupled to primary amines of peptides, and the ligand is then chelated to the rare earth metals Y and Tb. The tagged peptides are mixed and analyzed by LC-ESI-MS/MS. Peptides are quantified by measuring the relative signal intensities for the Y and Tb tag pairs in MS, which permits the quantitation of the original proteins generating the corresponding peptides. The protein is then identified by the corresponding peptide sequence from its MS/MS spectrum. The MECT method was evaluated by using standard proteins as model sample. The experimental results showed that metal chelate-tagged peptides chromatographically coeluted successfully during the reversed-phase LC analysis. The relative quantitation results were accurate for proteins using MECT. DTPA modification of the N-terminal of peptides promoted cleaner fragmentation (only y-series ions) in mass spectrometry and improved the confidence level of protein identification. The MECT strategy provides a simple, rapid, and economical alternative to current mass tagging technologies available.     

Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching

A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described. Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm. The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits. Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues. Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis. The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra. The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database. As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping.     

Quantitative measurements of cell-cell signaling peptides with single-cell MALDI MS

Cell-to-cell signaling peptides play important roles in neurotransmission, neuromodulation, and hormonal signaling. Significant progress has been achieved in qualitative investigations of signaling peptides in the nervous system using single cell matrix-assisted laser desorption/ionization mass spectrometry. However, quantitative information about signaling peptides is difficult to obtain with this approach because only small amounts of analytes are available for analysis. Here we describe several methods for quantitative microanalysis of peptides in individual Aplysia californica neurons and small pieces of tissue. Stable isotope labeling with d0- and d4-succinic anhydride and iTRAQ reagents has been successfully adopted for relative quantitation of nanoliter volume samples containing the Aplysia insulin C beta peptide. Comparative analysis of the C beta peptide release site, the upper labial nerve, and its synthesis location, the F- and C-clusters, shows that the release site possesses almost three times more of this compound. The method of standard addition permits absolute quantitation of the physiologically active neuropeptide cerebrin from small structures, including nerves and neuronal clusters, in the femtomole range with a limit of detection of 19 fmol. The simplicity of these methods and the commercial availability of the reagents allow quantitative measurements from a variety of small-volume biological samples.     

Non-gel-based dual 18O labeling quantitative proteomics strategy

To improve the quantitation of target proteins in proteomic analyses, we developed a non-gel-based, dual (18)O labeling strategy. This global isotope labeling method utilizes an acylating chemical reagent with two anhydride functional groups, bicyclic anhydride diethylenetriamine-N,N,N', N' ',N' '-pentaacetic acid (DTPA) dianhydride. In the first (18)O labeling method (chemical (18)O labeling) of our dual strategy, one functional group was covalently coupled to the primary amines of the peptides and (18)O from H2(18)O was incorporated at the other functional group by hydrolysis. In the second (18)O labeling method (chemical and enzyme-catalyzed (18)O labeling), chemical (18)O labeling and enzyme-catalyzed (18)O labeling of the carboxyl- termini of the peptides were combined. The acylation reaction between DTPA and the model peptides was rapid and specific, and the DTPA-modified N-termini of the peptides promoted only y-series ions in MS/MS. The two methods of (18)O labeling were accurate in the range 0.1-10 of (16)O/(18)O peptide ratios. The deviations of the methods were <20%. In contrast to current proteolytic (18)O labeling methods, there was no (18)O to (16)O back-exchange in the first method and no isotope peaks in MS in the second method. The combination of chemical and proteolytic (18)O labeling improved the confidence of the quantitation results.     

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.     

Dynamic collision-induced dissociation of peptides in a quadrupole ion trap mass spectrometer

The fragmentation of natural peptides using dynamic collision-induced dissociation (DCID), a novel fragmentation method for quadrupole ion traps, is demonstrated. Using leucine enkephalin as a diagnostic molecule, the fragmentation efficiencies and energetics of DCID are compared with other methods of collisional activation in ion traps such as conventional on-resonance excitation and high-amplitude short-time excitation (HASTE). A typical fragmentation efficiency of approximately 20% is achieved for DCID, which is significantly lower than conventional CID (maximum near 80%). Tandem mass spectra of two other peptides, substance P and oxidized insulin alpha-chain, demonstrate that product ion spectra for DCID are comparable to conventional or HASTE CID. Because DCID achieves fragmentation during the standard mass acquisition scan, no extra time is necessary for on-resonance excitation or product ion collection, so analysis times are reduced by a minimum of 10-15% depending on the scanning conditions. DCID therefore offers more tandem mass spectra per second than conventional methods of collisional activation, which could be highly advantageous for bottom-up proteomics separations.     

Quantification of cysteine oxidation in human estrogen receptor by mass spectrometry

Redox-dependent modifications of sulfhydryl groups within the two Cys4 zinc fingers of the estrogen receptor DNA-binding domain (ER-DBD) result in structural damage and loss of ER DNA-binding function, which parallels the situation observed in many ER-positive breast cancers. Quantitation of the redox status of cysteinyl thiols within ER-DBD employed cysteine-specific oxidants to induce varying degrees of oxidation in recombinant ER, followed by differential alkylation with the stable isotopic labeling reagents [12C2]-iodoacetic acid and [13C2]-bromoacetic acid. Subsequent proteolysis with LysC/Asp-N generated diagnostic peptides of which the C-terminal peptide of the second zinc finger is most strongly detected by mass spectrometry (MS) and serves as a suitable marker of ER-DBD redox status. Data were collected from two different MALDI-MS instruments: a time-of-flight and a linear ion trap (vMALDI-LIT). An analogous but larger synthetic peptide treated with three isotopic variants of the alkylating reagent modeled isotopic overlaps that might complicate the relative quantitation of cysteine oxidation. Despite the isotopic overlaps, excellent relative quantitation was achieved from MS data obtained from both instruments. This was also true of tandem MS/MS data from the vMALDI-LIT, which should facilitate selected reaction monitoring. Relative quantitation by MS also closely matched data from immunochemical methods.     

Peptide and protein quantitation by acid-catalyzed 18O-labeling of carboxyl groups

We have developed a new method that applies acidic catalysis with hydrochloric acid for (18)O-labeling of peptides at their carboxyl groups. With this method, peptides get labeled at their C-terminus, at Asp and Glu residues, and at carboxymethylated cysteine residues. Oxygen atoms at phosphate groups of phosphopeptide are not exchanged. Our elaborated labeling protocol is easy to perform, fast (5 h and 30 min), and results in 95-97 atom % incorporation of (18)O at carboxyl groups. Undesired side reactions, such as deamidation or peptide hydrolysis, occur only at a very low level under the conditions applied. In addition, data analysis can be performed automatically using common software tools, such as Mascot Distiller. We have demonstrated the capability of this method for the quantitation of peptides as well as for phosphopeptides.     

Polymeric activated ester reagents for off-line and on-line derivatizations of amine nucleophiles in high-performance liquid chromatography with ultraviolet and fluorescence detection

A novel polymeric activated ester reagent has been developed that improves final detectability and chromatographic performance in high-performance liquid chromatography (HPLC) for virtually all primary and secondary amines or amine analogues. This has involved the synthesis, characterization of final reagent, optimization of derivatization and separation conditions, and determination of analytical figures of merit. The polymeric reagent contained an activated ester linkage to the 9-fluorenyl group, which imparted ultraviolet (UV) and fluorescence (FL) detector properties to the final derivatives. Kinetic studies of these solid-phase (heterogeneous) reactions have been conducted, and specific rate constants were compared with those of the analogous solution reaction for the same substrates. Percent derivatizations have reached 90% and 70% for primary and secondary amines, respectively, under optimized conditions. High reaction reproducibility has been obtained by using the on-line approach, for more than 50 separate injections of the same amine substrate with a single solid-phase reactor. These solid-phase derivatizations have led to detection limits for typical amines in the low-parts-per-billion range. The final, overall methods can provide rapid, automatable, accurate, and precise detection and quantitation of primary/secondary amines and amine-like compounds in real-world sample matrices. As an illustrative example, amphetamine spiked in urine has been derivatized off-line and on-line, with minimum sample preparation, and detected via HPLC-UV/FL with acceptable accuracy and precision.     

Use of capillary zone electrophoresis to evaluate the binding of anionic carbohydrates to synthetic peptides derived from human serum amyloid P component.

Capillary zone electrophoresis was used to study interactions between anionic carbohydrates and synthetic peptides derived from the heparin-binding region of human serum amyloid P component. The method involves quantitation of unbound peptides after a charge-dependent electrophoretic separation of the peptide-carbohydrate mixture. The concentrations of free peptide were determined by extrapolating the obtained peak areas of the peptide in the presence of ligand to a standard curve. Dissociation constants in the 10(-5) M range were determined, and differences in binding affinity of various peptide modifications were illustrated. The assay requires minute amounts of material (sample volume is 7-15 nL), and as long as the reactants are soluble at the chosen conditions, no modifications or special characteristics of the interacting molecules are needed for their identification. It should be possible to use electrophoretic separation in capillaries to evaluate the binding of peptides to any ligand as long as the differences in charge/mass ratio between free and complexed peptide are of a sufficient magnitude as in the peptide-heparin binding demonstrated here.     

Improving de novo sequencing of peptides using a charged tag and C-terminal digestion

An improved method for peptide de novo sequencing by MALDI mass spectrometry is presented. The method couples a charge derivatization reaction with C-terminal digestion to modify tryptic peptides. The charge derivatization attaches a fixed charge group onto the N-termini of peptides, and the enzymatic digestion after the derivatization step removes C-terminal basic amino acid residues such as arginine and lysine. The fragmentation of the modified peptide(s) under low-energy CID conditions (MALDI Q-TOF mass spectrometer) yields a simplified yet complete ion series of the peptide sequence. The validity of the method is demonstrated by the results from several model protein digests, where peptide sequences were correctly deduced either manually or through an automated sequencing program.     

Identification of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling

A new method is presented to screen proteolytic mass maps of cross-linked protein complexes for the presence of cross-linked peptides and for the verification of proposed structures. On the basis of the incorporation of 18O from isotopically enriched water into the C-termini of proteolytic peptides, cross-linked peptides are readily distinguished in mass spectra by a characteristic 8 amu shift. This is due to the incorporation of two 18O atoms in each C-terminus, so that normal and surface-labeled peptides shift 4 amu and cross-linked peptides containing two C-termini will shift 8 amu compared with their unlabeled counterparts. The method is fast, sensitive, and reliable and can be combined with any available cross-linking reagent and a wide range of proteolytic agents. As proof of principle, we successfully applied the method to a complex of two DNA repair proteins (Rad18-Rad6) and identified the interaction domain.     

Mass spectral analysis of chloropicrin under negative ion chemical ionization conditions

A chemical ionization (CI) method is developed for the first time to obtain molecular weight information for chloropicrin (CP), which is used as a chemical warfare agent and as an insecticide. The study includes a detailed investigation on the behavior of CP under electron impact (EI) and CI. Reagent gases of different nature, i.e., methane, isobutane, ammonia, hydrogen, and carbon dioxide, were used for CI analysis. Negative ion mode is found more sensitive than positive ion mode for the EI/CI mass spectrometric analysis of CP, but none of the methods provided molecular weight information, except negative ion CI using ammonia as the reagent gas (NICI (NH3)). The NICI (NH3) showed formation of the quasi-molecular ion, [M + H]-, in addition to other adduct ions. The [M + H]- abundance critically depends on the source temperature, reagent gas pressure, and concentration of the analyte, and it can be 13% under optimized conditions by which CP can be confirmed unambiguously. This method meets the criteria used in official proficiency tests conducted by OPCW for confirming the molecular weight of the unknowns.     

Characterization of Enterobacteria using MALDI-TOF mass spectrometry

A method is proposed for the rapid classification of Gram-negative Enterobacteria using on-slide solubilization and trypsin digestion of proteins, followed by MALDI-TOF MS analysis. Peptides were identified from tryptic digests using microsequencing by tandem mass spectrometry and database searches. Proteins from the outer membrane family (OMP) were consistently identified in the Enterobacteria Escherichia coli, Enterobacter cloacae, Erwinia herbicola, and Salmonella typhimurium. Database searches indicate that these OMP peptides observed are unique to the Enterobacteria order.