Relative quantitation of peptides in wild-type and Cpe(fat/fat) mouse pituitary using stable isotopic tags and mass spectrometry

Cpe(fat/fat) mice have a point mutation in the coding region of the carboxypeptidase E gene that renders the enzyme inactive. As a result, these mice have reduced levels of several neuropeptides and greatly increased levels of the peptide processing intermediates that contain C-terminal basic residues. However, previous studies examined a relatively small number of neuropeptides. In the present study, we used a quantitative peptidomics approach with stable isotopic labels to examine the levels of pituitary peptides in Cpe(fat/fat) mice relative to wild-type mice. Pituitary extracts from mutant and wild type mice were labeled with the stable isotopic label [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propyl]trimethylammonium chloride containing nine atoms of hydrogen or deuterium. Then, the two samples were pooled and analyzed by liquid chromatography/mass spectrometry (LC/MS). The relative abundance of peptides was determined from a comparison of the intensities of the heavy and light peaks. Altogether, 72 peptides were detected in the Cpe(fat/fat) and/or wild-type mouse pituitary extracts of which 53 were identified by MS/MS sequencing. Several peptides identified in this analysis represent previously undescribed post-translational processing products of known pituitary prohormones. Of the 72 peptides detected in pituitary, 17 were detected only in the Cpe(fat/fat) mouse extracts; these represent peptide processing intermediates containing C-terminal basic residues. The peptides common to both Cpe(fat/fat) and wild-type mice were generally present at 2-5-fold lower levels in the Cpe(fat/fat) mouse pituitary extracts, although some peptides were present at equal levels and one peptide (acetyl beta-endorphin 1-31) was increased approximately 7-fold in the Cpe(fat/fat) pituitary extracts. In contrast, acetyl beta-endorphin 1-26 was present at approximately 10-fold lower levels in the Cpe(fat/fat) pituitary, compared with wild-type mice. The finding that many peptides are substantially decreased in Cpe(fat/fat) pituitary is consistent with the broad role for carboxypeptidase E in the biosynthesis of numerous neuropeptides.     

Design and synthesis of a solid-phase fluorescent mass tag

In this study, we demonstrate the design of a new solid-phase fluorescent mass tag (FMT) that contains the following features: (1) the FMT is synthesized using Fmoc chemistry which is simple, rapid, and cost-effective; (2) lysine is used as a uniformly labeled amino acid (using stable isotopes) to allow 8 Da difference between "heavy" and "light" tags; (3) a fluorescent molecule is coupled to the isotope tag that allows a tagged peptide to be detected by online fluorescence; and (4) an iodoacetyl reactive group provides cysteine reactivity. Using MALDI-TOF MS and HPLC, we show that the FMT reagent can be used to label standard cysteine-containing peptides as well as cysteine-containing peptides from a BSA tryptic digest.     

Trypsin catalyzed 16O-to-18O exchange for comparative proteomics: tandem mass spectrometry comparison using MALDI-TOF,ESI-QTOF,and ESI-ion trap mass spectrometers

Quantitative or comparative proteome analysis was initially performed with 2-dimensional gel electrophoresis with the inherent disadvantages of being biased towards certain proteins and being labor intensive. Alternative mass spectrometry-based approaches in conjunction with gel-free protein/peptide separation have been developed in recent years using various stable isotope labeling techniques. Common to all these techniques is the incorporation, biosynthetically or chemically, of a labeling moiety having either a natural isotope distribution of hydrogen, carbon, oxygen, or nitrogen (light form) or being enriched with heavy isotopes like deuterium, (13)C, (18)O, or (15)N, respectively. By mixing equal amounts of a control sample possessing for instance the light form of the label with a heavy-labeled case sample, differentially labeled peptides are detected by mass spectrometric methods and their intensities serve as a means for direct relative protein quantification. While each of the different labeling methods has its advantages and disadvantages, the endoprotease (16)O-to-(18)O catalyzed oxygen exchange at the C-terminal carboxylic acid is extremely promising because of the specificity assured by the enzymatic reaction and the labeling of essentially every protease-derived peptide. We show here that this methodology is applicable to complex biological samples such as a subfraction of human plasma. Furthermore, despite the relatively small mass difference of 4 Da between the two labeled forms, corresponding to the exchange of two oxygen atoms by two (18)O isotopes, it is possible to quantify differentially labeled proteins on an ion trap mass spectrometer with a mass resolution of about 2000 in automated data dependent LC-MS/MS acquisition mode. Post column sample deposition on a MALDI target parallel to on-line ESI-MS/MS enables the analysis of the same compounds by means of ESI- and MALDI-MS/MS. This has the potential to increase the confidence in the quantification results as well as to increase the sequence coverage of potentially interesting proteins by complementary peptide ionization techniques. Additionally the paired y-ion signals in tandem mass spectra of (16)O/(18)O-labeled peptide pairs provide a means to confirm automatic protein identification results or even to assist de novo sequencing of yet unknown proteins.     

RABA (Reductive Alkylation By Acetone): A novel stable isotope labeling approach for quantitative proteomics

Quantitative proteomics is challenging and various stable isotope based approaches have been developed to meet the challenge. Hereby we describe a simple, efficient, reliable, and inexpensive method named reductive alkylation by acetone (RABA) to introduce stable isotopes to peptides for quantitative analysis. The RABA method leads to alkylation of N-terminal and lysine amino groups with isopropyl moiety. Using unlabeled (d₀) and deuterium labeled (d₆) acetone, a 6 Da mass split is introduced to each isopropyl modification between the light and heavy isotope labeled peptides, which is ideally suited for quantitative analysis. The reaction specificity, stoichiometry, labeling efficiency, and linear range of the RABA method have been thoroughly evaluated in this study using standard peptides, tryptic digest of proteins, as well as human cell lysate. Reliable quantitative results have been consistently obtained in all experiments. We also applied the RABA method to quantitative analysis of proteins in spinal cords of transgenic mouse models of amyotrophic lateral sclerosis. Highly homologous proteins (transgenic human SOD1 and endogenous mouse SOD1) were distinguished and quantified using the method developed in this study. In addition, the quantitative results using the RABA approach were independently validated by Western blot.     

Selective identification and quantitative analysis of methionine containing peptides by charge derivatization and tandem mass spectrometry

To enable the development of a tandem mass spectrometry (MS/MS) based methodology for selective protein identification and differential quantitative analysis, a novel derivatization strategy is proposed, based on the formation of a "fixed-charge" sulfonium ion on the side-chain of a methionine amino acid residue contained within a protein or peptide of interest. The gas-phase fragmentation behavior of these side chain fixed charge sulfonium ion containing peptides is observed to result in exclusive loss of the derivatized side chain and the formation of a single characteristic product ion, independently of charge state or amino acid composition. Thus, fixed charge containing peptide ions may be selectively identified from complex mixtures, for example, by selective neutral loss scan mode MS/MS methods. Further structural interrogation of identified peptide ions may be achieved by subjecting the characteristic MS/MS product ion to multistage MS/MS (MS3) in a quadrupole ion trap mass spectrometer, or by energy resolved "pseudo" MS3 in a triple quadrupole mass spectrometer. The general principles underlying this fixed charge derivatization approach are demonstrated here by MS/MS, MS3 and "pseudo" MS3 analysis of side chain fixed-charge sulfonium ion derivatives of peptides containing methionine formed by reaction with phenacylbromide. Incorporation of "light" and "heavy" isotopically encoded labels into the fixed-charge derivatives facilitates the application of this method to the quantitative analysis of differential protein expression, via measurement of the relative abundances of the neutral loss product ions generated by dissociation of the light and heavy labeled peptide ions. This approach, termed "selective extraction of labeled entities by charge derivatization and tandem mass spectrometry" (SELECT), thereby offers the potential for significantly improved sensitivity and selectivity for the identification and quantitative analysis of peptides or proteins containing selected structural features, without requirement for extensive fractionation or otherwise enrichment from a complex mixture prior to analysis.     

Aggregation modes of the spin mono-labeled tylopeptin B and heptaibin peptaibiotics in frozen solutions of weak polarity as studied by PELDOR spectroscopy

The X band PELDOR spectroscopy was used to investigate the magnetic dipole-dipole interactions in glassy solutions of nitroxide mono-labeled tylopeptin B and heptaibin peptaibiotics at 77 K. Specifically, a study was performed of the tylopeptin B peptides labeled at either position 3, 8, or 13, denoted as T3, T8, and T13, respectively. The heptaibin analogs labeled at either position 2 or 14, denoted as H2 and H14, respectively, were also investigated. It was shown that in frozen glassy peptide solutions in methanol, the spin labels are randomly distributed over the solvent volume. This result points to the absence of specific dipolar interactions between the peptides under these conditions. However, peptide aggregation was detected in weakly polar methanol/toluene environments. To study the properties of the resulting aggregates, we examined the depth of modulation for the PELDOR traces as a function of the concentration of the peptides in solution and the distances between the spin labels in the aggregates. Based on the concentration dependencies, the number of peptide molecules in the aggregates was estimated. We find that this value ranges from 2 to 3, depending on the position of the spin label in the peptide sequence. The combined analysis of the distance spectra and the number of peptide molecules in the aggregates allows us to suggest that dimer formation is the prevailing mode of self-association. In the case of spin-labeled tylopeptin B, the molecules in the dimer are head-to-head oriented. In addition, the distance spectra of the aggregates show that the C-termini of the molecules in the tylopeptin B dimer are more mobile than the Ntermini. This phenomenon leads to an increase in the spread of the distances between the nitroxides as the label position is approaching the peptide C-terminus. For heptaibin, we show that two forms of dimerization (head-to-head and head-to-tail) occur. Finally, in addition to dimers, aggregates containing 3 or 4 peptide molecules, which give broad lines in the distance spectra, are seen in solution.     

Proteome analysis of camptothecin-treated cortical neurons using isotope-coded affinity tags

Isotope-coded affinity tags (ICATs) were employed to identify and quantitate changes in protein expression between control and camptothecin-treated mouse cortical neurons. Proteins extracted from control cortical neurons and those treated with camptothecin were labeled with the light and heavy isotopic versions of the ICAT reagents, respectively. ICAT-labeled samples were combined, proteolytically digested, and the derivatized peptides isolated using immobilized avidin chromatography. The peptides thus isolated were analyzed by reversed-phase liquid chromatography coupled directly to either a conventional ion-trap mass spectrometer (IT-MS) or a Fourier transform ion cyclotron resonance mass spectrometer (FTICR). While a majority of the peptide identifications were accomplished using IT-MS, FTICR was used to quantitate the relative abundances of the ICAT-labeled peptides taking advantage of its high resolution, sensitivity, and duty cycle. By using this combination of MS technologies we have thus far identified and quantified the expression of greater than 125 proteins from control and camptothecin-treated mouse cortical neurons. While proteins from most functional classes of proteins were identified, a particularly large percentage of the enzymes involved in glycolysis and the tricarboxylic acid cycle were observed.     

低浓度甲醛对多肽和蛋白化学修饰的质谱研究

采用基质辅助激光解析电离飞行时间质谱( MALDI-TOF MS)和纳升电喷雾四极杆飞行时间串联质谱( Nano-ESI -QTOF MS)技术,以标准肽段和流感病毒基质蛋白酶切肽段为模型,研究了甲醛对蛋白质和多肽主链的修饰作用。采用与实际病毒灭活过程一致的实验条件(4℃,0.025%(V/V)福尔马林(37%(w/w)甲醛溶液)处理72 h),进行甲醛与多肽的化学反应。结果表明,在实验条件下,甲醛能与标准肽段N端的氨基反应生成羟甲基加合物,再发生缩合反应生成亚胺,形成+12 Da的产物。此外,甲醛还能与标准肽段中的精氨酸、赖氨酸的侧链发生反应,生成+12 Da的反应产物。对流感病毒基质蛋白的酶切肽段与甲醛的反应的质谱分析结果显示,多数的肽段都生成了+24 Da的产物,质量的增加来源于肽段N端氨基(+12 Da)和C端精氨酸或赖氨酸的侧链(+12 Da)的贡献。此外,还观察到有一个漏切位点的肽段生成了+36 Da的产物。本研究结果表明,在实验条件下,低浓度甲醛主要与肽段和蛋白的N 端氨基,以及精氨酸和赖氨酸侧链发生反应。本研究为分析低浓度甲醛与蛋白质的反应产物提供了有效的质谱分析方法和解谱依据。     

Structural characterization of methionine and leucine enkephalins by hydrogen/deuterium exchange and electrospray ionization tandem mass spectrometry

Conformational changes in two endogenous opioid active pentapeptides methionine enkephalin (Met-enk) and leucine enkephalin (Leu-enk) induced by trifluoroethanol (TFE) were identified using hydrogen/deuterium exchange (HDX), coupled with electrospray ionization (ESI) mass spectrometry. The exchange features in individual amino acid residues were characterized by acquiring tandem mass spectra of the deuterated peptides. The exact identity of the labile hydrogens involved in HDX reveals that the monomer forms of both peptides adopt an unfolded conformation in aqueous solvent, but prefer the 5-->2 beta-turn secondary structure under the membrane-mimetic environment. The ESI mass spectra of Met-enk and Leu-enk also reveal that the dimer structure of these peptides coexists with the monomer conformation. The extent of the dimer structure is dependent on the peptide concentration and nature of the solvent. The non-polar solvents facilitate the dimer formation.     

Differential labeling of free and disulfide-bound thiol functions in proteins

A method for the simultaneous determination of the number of free cysteine groups and disulfide-bound cysteine groups in proteins has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. Liquid chromatography/electrochemistry/mass spectrometry was used to assign the N-(2-ferroceneethyl)maleimide (FEM) labeled free cysteine functionalities in a tryptic digest mixture, whereas a precursor ion scan enables the detection of peptides with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide (FMEA) labeled disulfide-bound cysteine groups after reduction. Fragment spectra of the labeled peptides yield an excellent coverage of b-type and y-type ions. The ferrocene labeled cysteines were fragmented as 412 Da (FEM) and 455 Da (FMEA). These fragment masses are significantly higher than unlabeled amino acids or dipeptides and are easily detected. The position of free and disulfide-bound cysteine may therefore be assigned in an amino acid sequence.     

Quantification of local hydration at the surface of biomolecules using dual-fluorescence labels

By using four labels of the 3-hydroxyflavone family displaying selective sensitivity to hydrogen bond (HB) donors and poor response to other polar molecules, we developed an approach for measuring local water concentration [H(2)O](L) (or partial volume of water: W(A) = [H(2)O](L)/55.6) in the label surrounding both in solvent mixtures and in biomolecules by the intensity ratio of two emissive forms of the label, N*/T*. Using a series of binary water/solvent mixtures with limited preferential solvation effects, a linear dependence of log(N*/T*) on the local concentration of HB donor was obtained and then used as a calibration curve for estimating the W(A) values in the surroundings of the probes conjugated to biomolecules. By this approach, we estimated the hydration of the labels in different peptides and their complexes with DNAs. We found that W(A) values for the label at the peptide N-terminus are lower (0.63-0.91) than for free labels and depend strongly on the nature of the N-terminal amino acid. When complexed with different DNAs, the estimated hydration of the labels conjugated to the labeled peptides was much lower (W(A) = 0-0.47) and depended on the DNA nature and linker-label structure. Thus, the elaborated method allows a site-specific evaluation of hydration at the surface of a biomolecule through the determination of the partial volume of water. We believe the developed procedure can be successfully applied for monitoring hydration at the surface of any biomolecule or nanostructure.     

Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation

Presently different opinions exist as to the degree of scrambling of amide hydrogens in gaseous protonated peptides and proteins upon collisional activation in tandem mass spectrometry experiments. This unsettled controversy is not trivial, since only a very low degree of scrambling is tolerable if collision-induced dissociation (CID) should provide reliable site-specific information from (1)H/(2)H exchange experiments. We have explored a series of unique, regioselectively deuterium-labeled peptides as model systems to probe for intramolecular amide hydrogen migration under low-energy collisional activation in an orthogonal quadrupole time-of-flight electrospray ionization (Q-TOF ESI) mass spectrometer. These peptides contain a C-terminal receptor-binding sequence and an N-terminal nonbinding region. When the peptides form a receptor complex, the amide hydrogens of the interacting sequences are protected against exchange with the solvent, while the amide hydrogens of the nonbinding sequences exchange rapidly with the solvent. We have utilized such long-lived complexes to generate peptides labeled with deuterium in either the binding or nonbinding region, and the expected regioselectivity of this labeling was confirmed after pepsin proteolysis. CID of such deuterated peptides, [M + 2H](2+), yielded fragment ions (b- and y-ions) having a deuterium content that resemble the theoretical values calculated for 100% scrambling. Thus, complete randomization of all hydrogen atoms attached to nitrogen and oxygen occurs in the gaseous peptide ion prior to its dissociation.     

Arginine-mimic labeling with guanidinoethanethiol to increase mass sensitivity of lysine-terminated phosphopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) generally shows better mass sensitivity for arginine-terminated peptides than for lysine-terminated peptides, presumed to arise from the higher proton affinity of the guanidine group in arginine. Here, we report a new method for analyzing phosphopeptides in which phosphopeptides are labeled with a novel chemical tag, guanidinoethanethiol (GET), by a beta-elimination/Michael addition before MS analysis. GET labeling converts phosphoserine into guanidinoethylcysteine (Gec) containing a guanidine moiety, along with an increase in mass of 21.1 Da. GET-labeled peptides are detected by MALDI MS with greatly increased peak intensities compared to those of intact phosphopeptides. In particular, GET labeling of lysine-terminated phosphopeptides dramatically increased peak intensity. GET labeling of lysine-terminated phosphopeptides improved sensitivity up to 22 times compared to that of the corresponding aminoethanethiol (AET) labeling, in which AET was used as a labeling tag containing an amino group instead of the guanidine group. These results show the guanidine group plays a very important role in increasing the observed sensitivity of MALDI MS for labeled peptide, derivatized from serine-phosphorylated peptides.     

ZoomQuant: An application for the quantitation of stable isotope labeled peptides

The main goal of comparative proteomics is the quantitation of the differences in abundance of many proteins between two different biological samples in a single experiment. By differentially labeling the peptides from the two samples and combining them in a single analysis, relative ratios of protein abundance can be accurately determined. Protease catalyzed (18)O exchange is a simple method to differentially label peptides, but the lack of robust software tools to analyze the data from mass spectra of (18)O labeled peptides generated by common ion trap mass spectrometers has been a limitation. ZoomQuant is a stand-alone computational tool that analyzes the mass spectra of (18)O labeled peptides from ion trap instruments and determines relative abundance ratios between two samples. Starting with a filtered list of candidate peptides that have been successfully identified by Sequest, ZoomQuant analyzes the isotopic forms of the peptides using high-resolution zoom scan spectrum data. The theoretical isotope distribution is determined from the peptide sequence and is used to deconvolute the peak areas associated with the unlabeled, partially labeled, and fully labeled species. The ratio between the labeled and unlabeled peptides is then calculated using several different methods. ZoomQuant's graphical user interface allows the user to view and adjust the parameters for peak calling and quantitation and select which peptides should contribute to the overall abundance ratio calculation. Finally, ZoomQuant generates a summary report of the relative abundance of the peptides identified in the two samples.     

Selective detection of thiosulfate-containing peptides using tandem mass spectrometry

Incubation of proteins or peptides containing disulfide bonds (S-S) with sodium sulfite (Na(2)SO(3)) cleaves S-S bonds producing approximately equimolar amounts of free thiols (-SH) and thiosulfates (-S-SO(3)H), a process known as sulfitolysis. Proteins and peptides containing thiosulfates were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by mass spectrometry (MS) and peptide mapping. The mass of the thiosulfate-containing peptide formed from oxidized insulin B chain was 3478.02 Da, 80 Da greater than the reduced peptide and corresponding precisely to addition of sulfur trioxide (SO(3)). Disulfide bond cleavage was also observed using RP-HPLC and MS after incubation of the intramolecular homodimer of mouse S100A8 (mass 20614 Da). The mass of HPLC-separated A8-SH was 10308 Da, and 10388 Da for A8-S-SO(3)H. Loss of SO(3) from multiply charged precursor ions was generally observed at elevated declustering potentials in the source region or within q(2) at relatively low collision energies (approximately 20 V). The characteristic loss of SO(3) at low collision energies preceded peptide backbone fragmentations at higher collision energies. Accurate mass measurement and charge-state discrimination, using a hybrid quadrupole time-of-flight mass spectrometer, allowed specific detection of thiosulfate-containing peptides. An information-dependent acquisition method, where the switch criterion was loss of m/z 79.9568, specifically identified 11 thiosulfate-containing peptides using nano-LC/MS from a tryptic digest of bovine serum albumin (BSA).     

Improving the precision of quantitative bottom-up proteomics based on stable isotope-labeled proteins

Stable isotope dilution-based quantitative proteomics with intact labeled proteins as internal standards in combination with a bottom-up approach, i.e., with quantification on the peptide level, is an established method. To explore the technical precision of this approach, calmodulin-like protein 3 was prepared in non-labeled (light) and SILAC-type labeled (heavy) form by cell-free synthesis, mixed, digested with trypsin, and analyzed by UPLC-ESI-MS. In total, 16 light/heavy peptide pair ratios were determined. Pair-wise comparison of ratios of 12 peptides selected according to S/N ratios >50 revealed that the majority exhibited ratios, which were different at a high level of statistical significance (p < 0.001). HPLC-MALDI-MS ratio data confirmed this observation, thus excluding the ionization method as a source of the observed ratio differences. Variation of the digestion time from 0.25 to 4 h showed that the light/heavy ratios of most peptides decrease with time, indicating a kinetic isotope effect leading to preferred cleavage of light calmodulin-like protein 3. The subset of peptides with statistically identical ratios resulted in an average ratio with a RSD of 1.0 %. The light/heavy ratio calculated on the basis of these peptides probably provides the most accurate molar protein ratio.     

An automated method for the analysis of stable isotope labeling data in proteomics

An algorithm is presented for the generation of a reliable peptide component peak table from liquid chromatography-mass spectrometry (LC-MS) and subsequent quantitative analysis of stable isotope coded peptide samples. The method uses chemical noise filtering, charge state fitting, and deisotoping toward improved analysis of complex peptide samples. Overlapping peptide signals in mass spectra were deconvoluted by correlation with modeled peptide isotopic peak profiles. Isotopic peak profiles for peptides were generated in silico from a protein database producing reference model distributions. Doublets of heavy and light labeled peak clusters were identified and compared to provide differential quantification of pairs of stable isotope coded peptides. Algorithms were evaluated using peptides from digests of a single protein and a seven-protein mixture that had been differentially coded with stable isotope labeling agents and mixed in known ratios. The experimental results correlated well with known mixing ratios.     

Dimethyl multiplexed labeling combined with microcolumn separation and MS analysis for time course study in proteomics

Stable-isotope labeling coupled with liquid-phase separation and MS analysis is a powerful technique for comparative proteomics. We developed a dimethyl labeling strategy (Anal. Chem. 2003, 75, 6843-6852 and J. Proteome Res. 2005, 4, 101-108) to label peptide N-terminus and epsilon-amino groups of Lys with water-soluble formaldehyde via reductive methylation, and an isotopic pair of formaldehyde is used for binary labeling on two sets of samples. In this study, this approach is extended to a four sample labeling by combining the binary isotopic reagents of formaldehyde (d0, d2) and the binary isotopic reducing reagents, sodium cyanoborohydride (d0, d3). To ensure sufficient mass difference, this multiplexed labeling is coupled with endoproteinase Lys-C instead of trypsin for digestion, resulting in at least two labeling sites with a mass difference of 4 Da for each pair of peptide digest. Moreover, multiplex dimethyl labeling was proved to have no significant isotopic effect during RP LC elution. This method was further applied for monitoring Lys-C digestion using hemoglobin as a model. Dimethyl labeled digests derived from seven time points (1-30 h) were grouped into two sets of sample mixtures, separated by nano-LC to reduce the complexity, and then analyzed by ESI-MS/MS. The temporal study reveals that Lys-C digestion was completed in 10-15 h for all detected peptides. The multiplex dimethyl method has not only provided a simultaneous detection mean for four sample sets but has also conserved all the advantages associated with the original binary method.     

Protected Amine Labels: A Versatile Molecular Scaffold for Multiplexed Nominal Mass and Sub-Da Isotopologue Quantitative Proteomic Reagents

We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (1) robust targeting of peptide N-termini and lysyl side chains, (2) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (3) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (4) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da, are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition, we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally, we provide exemplar data that extend the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers.