Diagnostic protein discovery using proteolytic peptide targeting and identification

Plasma protein profiling with mass spectrometry is currently being evaluated as a diagnostic tool for cancer and other diseases. These experiments consist of three steps: plasma protein fractionation, analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and comparisons of the MALDI profiles to develop diagnostic fingerprints using bioinformatic techniques. While preliminary results appear promising in small sample groups, the method is limited by the sensitivity of MALDI-MS for intact proteins, the limited mass range of MALDI-MS, and difficulties associated with isolating individual proteins for identification to validate the diagnostic fingerprint. Here we present an alternative and improved method directed toward diagnostic protein discovery, which incorporates proteolytic peptide profiling, bioinformatic targeting of ion signals, and MALDI tandem mass spectrometry (MS/MS) peptide sequencing, rather than fingerprinting. Pancreatic cancer patients, pancreatitis patients, and controls are used as the model system. Profiling peptides after enzymatic digestion improves sensitivity and extends the accessible protein molecular weight range when compared to intact protein profiling. The first step is to extract and fractionate the proteins from plasma. Each fraction is digested with trypsin and subsequently analyzed by MALDI-MS. Rather than using bioinformatic analysis as a pattern-matching technique, peptides are targeted based on the disease to control peak intensity ratios measured in the averages of all mass spectra in each group and t-tests of the intensity of each individual peak. The targeted peptide ion signals are subsequently identified using MALDI-MS/MS in quadrupole-TOF and tandem-TOF instruments. This study found not only the proteins targeted and identified by a previous protein profiling experiment, but also detected additional proteins. These initial results are consistent with the known biology of pancreatic cancer or pancreatitis, but are not specific to those diseases.     

'Information-Based-Acquisition' (IBA) technique with an ion-trap/time-of-flight mass spectrometer for high-throughput and reliable protein profiling

Highly complex protein mixtures can be analyzed after proteolysis using liquid chromatography/mass spectrometry (LC/MS). In an LC/MS run, intense peptide ions originating from high-abundance proteins are preferentially analyzed using tandem mass spectrometry (MS(2)), so obtaining the MS(2) spectra of peptide ions from low-abundance proteins is difficult even if such ions are detected. Furthermore, the MS(2) spectra may produce insufficient information to identify the peptides or proteins. To solve these problems, we have developed a real-time optimization technique for MS(2), called the Information-Based-Acquisition (IBA) system. In a preliminary LC/MS run, a few of the most intense ions detected in every MS spectrum are selected as precursors for MS(2) and their masses, charge states and retention times are automatically registered in an internal database. In the next run, a sample similar to that used in the first run is analyzed using database searching. Then, the ions registered in the database are excluded from the precursor ion selection to avoid duplicate MS(2) analyses. Furthermore, real-time de novo sequencing is performed just after obtaining the MS(2) spectrum, and an MS(3) spectrum is obtained for accurate peptide identification when the number of interpreted amino acids in the MS(2) spectrum is less than five. We applied the IBA system to a yeast cell lysate which is a typical crude sample, using a nanoLC/ion-trap time-of flight (IT/TOF) mass spectrometer, repeating the same LC/MS run five times. The obtained MS(2) and MS(3) spectra were analyzed by applying the Mascot (Matrix Science, Boston, MA, USA) search engine to identify proteins from the sequence database. The total number of identified proteins in five LC/MS runs was three times higher than that in the first run and the ion scores for peptide identification also significantly increased, by about 70%, when the MS(3) spectra were used, combined with the MS(2) spectra, before being subjected to Mascot analysis.     

A method for rapidly confirming protein N-terminal sequences by matrix-assisted laser desorption/ionization mass spectrometry

The N-terminal sequence is important for the identification of a protein and the confirmation of its N-terminal processing. Although mass spectrometry (MS) is a sensitive and high-throughput method to sequence and identify peptides and proteins, N-terminal peptides, diluted among most of the peptides that do not originate at the N-termini, are not easy to identify directly with MS. To develop a simple and rapid method to identify and sequence the N-terminal peptide of a protein, a new strategy based on specific sulfonation of terminal amino groups and selective monitoring of the sulfonated peptide was introduced. After a protein had been guanidinated, 2-sulfobenzoylated, and reduced, it was digested with trypsin and analyzed by MS. Because of the strong acidity of sulfonic groups and the specific sulfonation of alpha-amino groups, the sulfonated N-terminal peptide dominated as base peak in the negative mode peptide mass fingerprint (PMF) and was easy to identify. The N-terminal peptide was then selected as precursor ion for tandem mass spectrometric (MS/MS) analysis. Four proteins were tested with this method and their N-terminal peptides were successfully recognized and sequenced. The results suggest that the addition of a sulfonic acid group facilitates the identification and de novo sequencing of N-terminal peptides.     

Continuous sample deposition from reversed-phase liquid chromatography to tracks on a matrix-assisted laser desorption/ionization precoated target for the analysis of protein digests

Peptide mass fingerprinting by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) is one of the standard high-throughput methods for protein identification today. Traditionally this method has been based on spotting peptide mixtures onto MALDI targets. While this method works well for more abundant proteins, low-abundance proteins mixed with high-abundance proteins tend to go undetected due to ion suppression effects, instrumental dynamic range limitations and chemical noise interference. We present an alternative approach where liquid chromatography (LC) effluent is continuously collected as linear tracks on a MALDI target. In this manner the chromatographic separation is spatially preserved on the target, which enables generation of off-line LC-MS and LC-MS/MS data by MALDI. LC-MALDI sample collection provides improved sensitivity and dynamic range, spatial resolution of peptides along the sample track, and permits peptide mass mapping of low-abundance proteins in mixtures containing high-abundance proteins. In this work, standard and ribosomal protein digests are resolved and captured using LC-MALDI sample collection and analyzed by MALDI-TOF-MS.     

Chemically-assisted fragmentation combined with multi-dimensional liquid chromatography and matrix-assisted laser desorption/ionization post source decay, matrix-assisted laser desorption/ionization tandem time-of flight or matrix-assisted laser desorption/ionization tandem mass spectrometry for improved sequencing of tryptic peptides

Derivatization of tryptic peptides using an Ettan CAF matrix-assisted laser desorption/ionization (MALDI) sequencing kit in combination with MALDI-post source decay (PSD) is a fast, accurate and convenient way to obtain de novo or confirmative peptide sequencing data. CAF (chemically assisted fragmentation) is based on solid-phase derivatization using a new class of water stable sulfonation agents, which strongly improves PSD analysis and simplifies the interpretation of acquired spectra. The derivatization is performed on solid supports, ZipTip(microC18, limiting the maximum peptide amount to 5 microg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 microg of protein. To increase the number of peptides that could be sequenced, derivatized peptides were purified using multidimensional liquid chromatography (MDLC) prior to MALDI sequencing. Following the first dimension strong cation exchange (SCX) chromatography step, modified peptides were separated using reversed-phase chromatography (RPC). During the SCX clean up step, positively charged peptides are retained on the column while properly CAF-derivatized peptides (uncharged) are not. A moderately complex tryptic digest, prepared from six different proteins of equimolar amounts, was CAF-derivatized and purified by MDLC. Fractions from the second dimension nano RPC step were automatically sampled and on-line dispensed to MALDI sample plates and analyzed using MALDI mass spectrometry fragmentation techniques. All proteins in the derivatized protein mixture digest were readily identified using MALDI-PSD or MALDI tandem mass spectrometry (MS/MS). More than 40 peptides were unambiguously sequenced, representing a seven-fold increase in the number of sequenced peptides in comparison to when the CAF-derivatized protein mix digest was analyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion, MDLC purification of CAF-derivatized peptides significantly increases the success rate for de novo and confirmative sequencing using various MALDI fragmentation techniques. This new approach is not only applicable to single protein digests but also to more complex digests and could, thus, be an alternative to electrospray ionization MS/MS for peptide sequencing.     

Fully automated chip-based nanoelectrospray combined with electron transfer dissociation for high throughput top-down proteomics

The conventional protocol for protein identification by electrospray ionization mass spectrometry (MS) is based on enzymatic digestion which renders peptides to be analyzed by liquid chromatography-MS and collision-induced dissociation (CID) multistage MS, in the so-called bottom-up approach. Though this method has brought a significant progress to the field, many limitations, among which, the low throughput and impossibility to characterize in detail posttranslational modifications in terms of site(s) and structure, were reported. Therefore, the research is presently focused on the development of procedures for efficient top-down fragmentation of intact protein ions. In this context, we developed here an approach combining fully automated chip-based-nanoelectrospray ionisation (nanoESI), performed on a NanoMate robot, with electron transfer dissociation (ETD) for peptide and top-down protein sequencing and identification. This advanced analytical platform, integrating robotics, microfluidics technology, ETD and alternate ETD/CID, was tested and found ideally suitable for structural investigation of peptides and modified/functionalized peptides as well as for top-down analysis of medium size proteins by tandem MS experiments of significantly increased throughput and sensitivity. The obtained results indicate that NanoMate-ETD and ETD/CID may represent a viable alternative to the current MS strategies, with potential to develop into a method of routine use for high throughput top-down proteomics.     

An algorithm for interpretation of low-energy collision-induced dissociation product ion spectra for de novo sequencing of peptides

An algorithm for interpretation of product ion spectra of peptides generated from ion trap mass spectrometry is developed for de novo amino acid sequencing of peptides for the purpose of protein identification. It is based on a multi-pass analysis of product ion data using a rigorous data extraction and sequence interpretation protocol in the initial pass. The extraction/interpretation algorithm becomes more relaxed in subsequent passes, considering more of the fragment ions, and potentially more sequence candidates. The possible peptide sequences generated by the algorithm are scored according to those sequences which best explain the fragment ion spectrum. These sequences are searched against a protein database using a BLAST search engine to find likely protein candidates. The method is also suitable for locating and determining protein modifications, and can be applied to de novo interpretation of peptide fragment ions in the tandem mass (MS/MS) spectrum produced from a mixture of two peptides having similar nominal mass, but different sequences. Using a known protein, bovine serum albumin, as an example, it is illustrated that this method is rapid and efficient for MS/MS spectral interpretation. This method combined with BLAST programs is then applied to search homologies and to generate information on post-translational modifications of an unknown protein isolated from shark cartilage that does not have a complete genome or proteome database.     

De novo analysis of protein N-terminal sequence utilizing MALDI signal enhancing derivatization with Br signature

De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.     

Edman降解中异硫氰酸苯酯新修饰位点的发现和验证

Edman降解是最早建立的一种用于多肽和蛋白质氨基端测序的方法,该方法现在仍被广泛用于生物化学领域。随着高通量蛋白质组学技术的发展和应用,该方法中的异硫氰酸苯酯反应被用于修饰蛋白质氨基端,并用于检测蛋白质水解位点。但还没有异硫氰酸苯酯是否可以修饰其他氨基酸侧链并影响多肽序列分析的研究。为了探究其修饰其他氨基酸的可能性,本文利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和液相色谱-串联质谱（LC-MS/MS）研究了异硫氰酸苯酯对一个模型肽的化学修饰。质谱数据解析后发现在高浓度异硫氰酸苯酯的反应条件下,组氨酸上可以引入一个新的异硫氰酸苯酯修饰位点。这一修饰位点的发现预示着通过改变实验条件或分析方法,可以更准确地利用Edman降解和蛋白质组学技术分析多肽和蛋白质。     

New approach for rapid detection of known hemoglobin variants using LC-MS/MS combined with a peptide database

The identification of hemoglobin (Hb) variants is usually performed by means of different analytical steps and methodologies. Phenotypic methods, such as gel electrophoresis and high performance liquid chromatography, are used to detect the different electrophoretic or chromatographic behaviors of hemoglobin variants in comparison to HbA0 used as a control. These data often need to be combined with mass spectrometry analyses of intact globins and their tryptic peptide mixtures. As an alternative to a 'step-by-step' procedure, we have developed a 'single step' approach for the identification of Hb variants present in biological samples. This is based on the microHPLC-ESI-MS/MS analysis of the peptide mixture generated by a tryptic digestion of diluted Hb samples and an in-house new database containing solely the variant tryptic peptide of known human Hb variants. The experimental results (full MS and MS/MS spectra) are correlated with theoretical mass spectra generated from our in-house-built variant peptide database (Hbp) using the SEQUEST algorithm. Simple preparation of samples and an automated identification of the variant peptide are the main characteristics of this approach, making it an attractive method for the detection of Hb variants at the routine clinical level. We have analyzed 16 different samples, each containing a different known variant of hemoglobin.     

Feasibility of SU-8-based capillary electrophoresis-electrospray ionization mass spectrometry microfluidic chips for the analysis of human cell lysates

Monolithically integrated, polymer (SU-8) microchips comprising an electrophoretic separation unit, a sheath flow interface and an ESI emitter were developed to improve the speed and throughput of proteomics analyses. Validation of the microchip method was performed based on peptide mass fingerprinting and single peptide sequencing of selected protein standards. Rapid, yet reliable identification of four biologically important proteins (cytochrome C, β-lactoglobulin, ovalbumin and BSA) confirmed the applicability of the SU-8 microchips to ambitious proteomic applications and allowed their use in the analysis of human muscle cell lysates. The characteristic tryptic peptides were easily separated with plate numbers approaching 10(6), and with peak widths at half height as low as 0.6 s. The on-chip sheath flow interface was also exploited to the introduction of an internal mass calibrant along with the sheath liquid which enabled accurate mass measurements by high-resolution Q-TOF MS. Additionally, peptide structural characterization and protein identification based on MS/MS fragmentation data of a single tryptic peptide was obtained using an ion trap instrument. Protein sequence coverages exceeding 50% were routinely obtained without any pretreatment of the proteolytic samples and a typical total analysis time from sampling to detection was well below ten minutes. In conclusion, monolithically integrated, dead-volume-free, SU-8 microchips proved to be a promising platform for fast and reliable analysis of complex proteomic samples. Good analytical performance of the microchips was shown by performing both peptide mass fingerprinting of complex cell lysates and protein identification based on single peptide sequencing.     

Strategies in protein sequencing and characterization: Multi-enzyme digestion coupled with alternate CID/ETD tandem mass spectrometry

A strategy based on a simultaneous multi-enzyme digestion coupled with electron transfer dissociation (ETD) and collision-induced dissociation (CID) was developed for protein sequencing and characterization, as a valid alternative platform in ion-trap based proteomics. The effect of different proteolytic procedures using chymotrypsin, trypsin, a combination of both, and Lys-C, was carefully evaluated in terms of number of identified peptides, protein coverage, and score distribution. A systematic comparison between CID and ETD is shown for the analysis of peptides originating from the in-solution digestion of standard caseins. The best results were achieved with a trypsin/chymotrypsin mix combined with CID and ETD operating in alternating mode. A post-database search validation of MS/MS dataset was performed, then, the matched peptides were cross checked by the evaluation of ion scores, rank, number of experimental product ions, and their relative abundances in the MS/MS spectrum. By integrated CID/ETD experiments, high quality-spectra have been obtained, thus allowing a confirmation of spectral information and an increase of accuracy in peptide sequence assignments. Overlapping peptides, produced throughout the proteins, reduce the ambiguity in mapping modifications between natural variants and animal species, and allow the characterization of post translational modifications. The advantages of using the enzymatic mix trypsin/chymotrypsin were confirmed by the nanoLC and CID/ETD tandem mass spectrometry of goat milk proteins, previously separated by two-dimensional gel electrophoresis.     

A Negative Ion Mass Spectrometry Approach to Identify Cross-Linked Peptides Utilizing Characteristic Disulfide Fragmentations

Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS(3) analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies.     

Automatic internal calibration in liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry of protein digests

Accurately measured peptide masses can be used for large-scale protein identification from bacterial whole-cell digests as an alternative to tandem mass spectrometry (MS/MS) provided mass measurement errors of a few parts-per-million (ppm) are obtained. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) routinely achieves such mass accuracy either with internal calibration or by regulating the charge in the analyzer cell. We have developed a novel and automated method for internal calibration of liquid chromatography (LC)/FTICR data from whole-cell digests using peptides in the sample identified by concurrent MS/MS together with ambient polydimethylcyclosiloxanes as internal calibrants in the mass spectra. The method reduced mass measurement error from 4.3 +/- 3.7 ppm to 0.3 +/- 2.3 ppm in an E. coli LC/FTICR dataset of 1000 MS and MS/MS spectra and is applicable to all analyses of complex protein digests by FTICRMS.     

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Simple and efficient digestion of proteins, particularly hydrophobic membrane proteins, is of significance for comprehensive proteome analysis using the bottom-up approach. We report a microwave-assisted acid hydrolysis (MAAH) method for rapid protein degradation for peptide mass mapping and tandem mass spectrometric analysis of peptides for protein identification. It uses 25% trifluoroacetic acid (TFA) aqueous solution to dissolve or suspend proteins, followed by microwave irradiation for 10 min. This detergent-free method generates peptide mixtures that can be directly analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) without the need of extensive sample cleanup. LC-MALDI MS/MS analysis of the hydrolysate from 5 microg of a model transmembrane protein, bacteriorhodopsin, resulted in almost complete sequence coverage by the peptides detected, including the identification of two posttranslational modification sites. Cleavage of peptide bonds inside all seven transmembrane domains took place, generating peptides of sizes amenable to MS/MS to determine possible sequence errors or modifications within these domains. Cleavage specificity, such as glycine residue cleavage, was observed. Terminal peptides were found to be present in relatively high abundance in the hydrolysate, particularly when low concentrations of proteins were used for MAAH. It was shown that these peptides could still be detected from MAAH of bacteriorhodopsin at a protein concentration of 1 ng/microl or 37 fmol/microl. To evaluate the general applicability of this method, it was applied to identify proteins from a membrane protein enriched fraction of cell lysates of human breast cancer cell line MCF7. With one-dimensional LC-MALDI MS/MS, a total of 119 proteins, including 41 membrane-associated or membrane proteins containing one to 12 transmembrane domains, were identified by MS/MS database searching based on matches of at least two peptides to a protein.     

Direct ampholyte-free liquid-phase isoelectric peptide focusing: application to the human serum proteome

In this study, we utilized a multidimensional peptide separation strategy combined with tandem mass spectrometry (MS/MS) for the identification of proteins in human serum. After enzymatically digesting serum with trypsin, the peptides were fractionated using liquid-phase isoelectric focusing (IEF) in a novel ampholyte-free format. Twenty IEF fractions were collected and analyzed by reversed-phase microcapillary liquid chromatography (microLC)-MS/MS. Bioinformatic analysis of the raw MS/MS spectra resulted in the identification of 844 unique peptides, corresponding to 437 proteins. This study demonstrates the efficacy of ampholyte-free peptide autofocusing, which alleviates peptide losses in ampholyte removal strategies. The results show that the separation strategy is effective for high-throughput characterization of proteins from complex proteomic mixtures.     

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.     

Towards a human repertoire of monocytic lysosomal proteins

The lysosomal compartment of human monocytic cells has never been investigated by a proteomic approach. By a combination of one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis, protein identification by N-terminal sequencing, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting and tandem mass spectrometry (MS/MS) peptide sequence analysis, we initiated an exhaustive study of the human lyososomal proteome, which aims at establishing a 2-D reference map of human soluble lyososomal proteins. Human monocytic U937 cells were induced to secrete lysosomal soluble hydrolases by addition of NH4Cl in the culture medium. Since lysosomal soluble proteins are characterized by the presence of mannose-6-phosphate, they were purified on an affinity support bearing mannose-6-phosphate receptor. Analysis of the purified fraction led to the preliminary identification of fifteen proteins, among which twelve are well-known lysosomal hydrolases, one is assumed to be lysosomal on the basis of sequence homology to cysteine proteinases of the papain family, and two (leukocystatin and the human cellular repressor of E1A-stimulated genes) are described here for the first time as mannose-6-phosphate-containing proteins.     

Identification of proteins separated by one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis with matrix-assisted laser desorption/ionization ion trap mass spectrometry; comparison with matrix-assisted laser desorption/ionization time-of-flight mass fingerprinting

Digests from ten gel bands containing low abundance proteins were analyzed by both matrix-assisted laser desorption/ionization ion trap (MALDI-IT) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) methods. MALDI-TOF techniques were able to identify only one protein from all 10 gel bands, while MALDI-IT identified eight proteins from the same 10 bands. The ability to perform MS/MS experiments with a MALDI-IT instrument leads to protein identifications based on both peptide molecular mass and sequence information, and is much less prone to errors and uncertainties introduced by peptide fingerprinting methodologies in which protein identification is based on peptide molecular masses alone.     

Cleavable hydrophilic linker for one-bead-one-compound sequencing of oligomer libraries by tandem mass spectrometry

We have developed a method for the rapid and unambiguous identification of sequences of hit compounds from one-bead-one-compound combinatorial libraries of peptide and peptoid ligands. The approach uses a cleavable linker that is hydrophilic to help reduce nonspecific binding to biological samples and allows for the attachment of a halogen tag, which greatly facilitates post-screening sequencing by tandem mass spectrometry (MS/MS). The linker is based on a tartaric acid unit, which, upon cleavage from resin, generates a C-terminal aldehyde. This aldehyde can then be derivatized with a bromine-containing amino-oxy compound that serves as an isotope tag for subsequent MS/MS analysis of y-ion fragments. We have applied this linker and method to the syntheses of a number of peptoids that vary in sequence and length and have also demonstrated single-bead sequencing of a peptoid pentamer. The linker is also shown to have very low levels of nonspecific binding to proteins.