Charge derivatization by 4-sulfophenyl isothiocyanate enhances peptide sequencing by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

High-sensitivity, rapid identification of proteins in proteomic studies normally uses a combination of one- or two-dimensional electrophoresis together with mass spectrometry. The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have increased its application in recent years. The most common method of 'peptide fingerprinting' often may not provide robust identification. Normally additional sequence information by post-source decay (PSD) MALDI-TOFMS provides additional constraints for database searches to achieve highly confident results. Here we describe a derivatization procedure to facilitate the acquisition of such sequence information. Peptide digests from a skin-expressed protein were modified with 4-sulfophenyl isothiocyanate. The resulting peptides carry a fixed negative charge at the N-terminal end and the resulting PSD spectrum is dominated by C-terminal y-type ions. The sequence information in most cases can be obtained manually or with simple programming tools. Methods of optimizing the procedure and increasing the sensitivity are discussed.     

Sequit: software for de novo peptide sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry

Peptide sequencing by mass spectrometry is gaining increasing importance for peptide chemistry and proteomics. However, available tools for interpreting matrix-assisted laser desorption/ionization post-source decay (MALDI-PSD) mass spectra depend on databases, and identify peptides by matching experimental data with spectra calculated from database sequences. This severely obstructs the identification of proteins and peptides not listed in databases or of variations, e.g. mutated proteins. The development of a new computer program for database-independent peptide sequencing by MALDI-PSD mass spectrometry is reported here. This computer program was validated by the determination of the correct sequences for various peptides including sequences listed in the sequence databases, but also for peptides that deviate from database sequences or are completely artificial. This strategy should substantially facilitate the identification of novel or variant peptides and proteins, and increase the power of MALDI-PSD analyses in proteomics.     

Fragmentation and sequencing of cyclic peptides by matrix-assisted laser desorption/ionization post-source decay mass spectrometry.

A series of synthetic cyclic decapeptides and other smaller cyclic peptides were analyzed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The investigated compounds were cyclized in a head-to-tail manner and contained non-proteinaceous amino acids, such as D-phenylalanine, D,L-4-carboxyphenylalanine, epsilon-aminocaproic acid, and gamma-aminobutyric acid, and were synthesized in a program to develop inhibitors of pp60(c-src) (Src), a tyrosine kinase that is involved in signal transduction and growth regulation. Post-source decay (PSD) spectra of the cyclic peptides featured abundant sequence ions. Two preferential ring opening reactions were detected resulting in linear fragment ions with an N-terminus of proline and a C-terminus of glutamic acid, respectively. MALDI-PSD spectra even permitted de novo sequencing of some cyclic peptides. Systematic studies on cyclic peptides using this method of fragmentation have not been reported to date. This work presents an easy mass spectrometric method, MALDI-PSD, for the characterization and identification of cyclic peptides.     

Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry

Guanidination of the epsilon-amino group of lysine-terminated tryptic peptides can be accomplished selectively in one step with O-methylisourea hydrogen sulfate. This reaction converts lysine residues into more basic homoarginine residues. It also protects the epsilon-amino groups against unwanted reaction with sulfonation reagents, which can then be used to selectively modify the N-termini of tryptic peptides. The combined reactions convert lysine-terminated tryptic peptides into modified peptides that are suitable for de novo sequencing by postsource decay matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The guanidination reaction is very pH dependent. Product yields and reaction kinetics were studied in aqueous solution using either NaOH or diisopropylethylamine as the base. Methods are reported for derivatizing and sequencing lysine-terminated tryptic peptides at low pmole levels. The postsource decay (PSD) MALDI tandem mass spectra of a model peptide (VGGYGYGAK), the homoarginine analog and the sulfonated homoarginine analog are compared. These spectra show the influence that each chemical modification has on the peptide fragmentation pattern. Finally, we demonstrate that definitive protein identifications can be achieved by PSD MALDI sequencing of derivatized peptides obtained from solution digests of model proteins and from in-gel digests of 2D-gel separated proteins.     

Solid-phase derivatization of tryptic peptides for rapid protein identification by matrix-assisted laser desorption/ionization mass spectrometry

Solid-phase sulfonation of tryptic peptides adsorbed to C18 muZipTips has been carried out to facilitate de novo sequencing with mass spectrometry. Peptides are reacted with the sulfonation reagent while they are still adsorbed to the solid phase. Excess reagent passes through the ZipTip to waste. Washing the products before subsequent elution from the mini-column also affords sample cleanup prior to analysis. Near quantitative N-terminal sulfonation can be achieved reliably at room temperature in only a few seconds. The method has been applied successfully to model peptides and to solution or in-gel digests of proteins. Current sequencing limits are about 100 fmol of protein. Multiplexed sample sulfonation reactions have been carried out with a manual 8-position micropipettor or using centrifugal force to reliably pass reagents and wash solutions over sample-loaded ZipTips. With multiplexing, overall preparation times have been reduced to about 1 min per sample. The solid-phase format facilitates efficient use of precious digest samples by enabling them to be recovered from the matrix-assisted laser desorption/ionization (MALDI) sample stage after mass fingerprinting, derivatized and re-analyzed by MALDI postsource decay mass spectrometry.     

Structural analysis of phosphatidylcholines by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The utility of post-source decay (PSD) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was investigated for the structural analysis of phosphatidylcholine (PC). PC did not produce detectable negative molecular ion from MALDI, but positive ions were observed as both [PC+H](+) and [PC+Na](+). The PSD spectra of the protonated PC species contained only one fragment corresponding to the head group (m/z 184), while the sodiated precursors produced many fragment ions, including those derived from the loss of fatty acids. The loss of fatty acid from the C-1 position (sn-1) of the glycerol backbone was favored over the loss of fatty acid from the C-2 position (sn-2). Ions emanating from the fragmentation of the head group (phosphocholine) included [PC+Na-59](+), [PC+Na-183](+) and [PC+Na-205](+), which corresponded to the loss of trimethylamine (TMA), non-sodiated choline phosphate and sodiated choline phosphate, respectively. Other fragments reflecting the structure of the head group were observed at m/z 183, 146 and 86. The difference in the fragmentation patterns for the PSD of [PC+Na](+) compared to [PC+H](+) is attributed to difference in the binding of Na(+) and H(+). While the proton binds to a negatively charged oxygen of the phosphate group, the sodium ion can be associated with several regions of the PC molecule. Hence, in the sodiated PC, intermolecular interaction of the negatively charged oxygen of the phosphate group, along with sodium association at multiple sites, can lead to a complex and characteristic ion fragmentation pattern. The preferential loss of sn-1 fatty acid group could be explained by the formation of an energetically favorable six-member ring intermediate, as apposed to the five-member ring intermediate formed prior to the loss of sn-2 fatty acid group.     

Sodium-tolerant matrix for matrix-assisted laser desorption/ionization mass spectrometry and post-source decay of oligonucleotides

A mixture of 2',4',6'-trihydroxyacetophenone in acetonitrile and aqueous triammonium citrate solution in a 1:1 molar proportion (0.2 M concentration) was found to be a good matrix for the detection of synthetic oligodeoxynucleotide samples. A high proportion of volatile solvent as well as the high salt content ensure fast co-crystallization of the matrix, co-matrix and analyte molecules. Matrix-assisted laser desorption/ionization (MALDI) mass spectra obtained in negative ion reflectron mode from samples prepared with this protocol show deprotonated molecules [M - H](-), rather than sodium adducts, as the most abundant ions even when up to 50 mM of sodium chloride is present in the sample. The matrix is shown to be effective for low mass modified single nucleotides as well as for longer oligodeoxynucleotides (up to 18mer). Post-source decay (PSD) mass spectra can also be obtained by increasing the laser fluence. Simple sequence information such as the identity and localization of a deleted base or the 5'/3' orientation can then easily be obtained. The calibration method and mass accuracy required are discussed depending on the type of information required.     

Improved procedures for N-terminal sulfonation of peptides for matrix-assisted laser desorption/ionization post-source decay peptide sequencing

Post source decay (PSD) analysis of precursor ions generated from matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is a powerful tool for amino acid sequencing and primary structure analysis of proteins. N-Terminal sulfonation has become an effective derivatization strategy in facilitating de novo peptide sequencing by the formation of predominate y-type ion series in MALDI PSD spectra. Recently, an effective and inexpensive N-terminal derivatization method has been reported using 4-sulfophenyl isothiocyanate (SPITC) as the derivatization reagent (J. Mass. Spectrom. 2003; 38: 373-377). In this paper, we report an improvement in the derivatization procedure with this reagent that involves replacing an organic co-reagent with other chemicals and eliminating the use of organic solvent. The method is demonstrated on a model peptide and on tryptic digests of two proteins. The results indicate that the improved sulfonation reaction can be implemented with high efficiency under aqueous conditions and that the sensitivity of mass detection can be increased considerably.     

Structure analysis of branched oligosaccharides using post-source decay in matrix-assisted laser desorption ionization mass spectrometry

Post-source decay matrix-assisted laser desorption ionization (PSD-MALDI) of sodium ion-attached branched oligosaccharides derived from glycoproteins was demonstrated as a method of structure analysis by reflectron time-of-flight (TOF) mass spectrometry. Mono-, di- and triantennary structures were investigated. The fragmentation patterns of these (structurally related) substances as obtained in the positive-ion mode showed characteristic differences correlated with branching sites and linkage positions. Two-bond ring cleavages as known from fast atom bombardment/collision-induced dissociation and IR laser desorption mass spectrometry were also observed. Internal fragment ions formed by up to four consecutive cleavages were obtained with high intensity, allowing the branching structure of complex carbohydrates to be identified. PSD-MALDI of oligosaccharides is characterized by high sensitivity, very good signal-to-noise ratios and high reproducibility of fragmentation patterns and signal intensities.     

Protein identification by accurate mass matrix-assisted laser desorption/ionization imaging of tryptic peptides

The spatial distribution of proteins in tissue sections can be used to identify potential markers for pathological processes. Tissue sections are often subjected to enzymatic digestion before matrix‐assisted laser desorption/ionization (MALDI) imaging. This study is targeted at improving the on‐tissue identification of tryptic peptides by accurate mass measurements and complementary off‐line liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) analysis. Two adjacent mouse brain sections were analyzed in parallel. The first section was spotted with trypsin and analyzed by MALDI imaging. Direct on‐tissue MS/MS experiments of this section resulted in the identification of 14 peptides (originating from 4 proteins). The second tissue section was homogenized, fractionated by ultracentrifugation and digested with trypsin prior to LC/ESI‐MS/MS analysis. The number of identified peptides was increased to 153 (corresponding to 106 proteins) by matching imaged mass peaks to peptides which were identified in these LC/ESI‐MS/MS experiments. All results (including MALDI imaging data) were based on accurate mass measurements (RMS <2 ppm) and allow a confident identification of tryptic peptides. Measurements based on lower accuracy would have led to ambiguous or misleading results. MS images of identified peptides were generated with a bin width (mass range used for image generation) of Δm/z = 0.01. The application of accurate mass measurements and additional LC/MS measurements increased both the quality and the number of peptide identifications. The advantages of this approach for the analysis of biological tissue sections are demonstrated and discussed in detail. Results indicate that accurate mass measurements are needed for confident identification and specific image generation of tryptic peptides in tissue sections. Copyright © 2011 John Wiley & Sons, Ltd.     

Evaluation of charge derivatization of a proteolytic protein digest for improved mass spectrometric analysis: de novo sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry.

A simple mass spectrometric method to sequence a recombinant phosphoenolpyruvate carboxykinase of known structure and a novel variant of unknown structure isolated from Anaerobiospirillum succiniciproducens and Actinobacillus succinogenes 130Z, respectively, was evaluated. The proteolytic digests of the proteins were each chemically derivatized at the N-terminus by addition of a tris(trimethoxyphenyl)phosphoniumacetyl (TMPP(+)-Ac) group to produce peptides with a fixed positive charge. The derivatized digests were then partially separated by reversed-phase high-performance liquid chromatography. The fractions collected were subjected to matrix-assisted laser desorption/ionization post-source decay (MALDI/PSD) mass spectrometric analysis. The resulting spectra are sufficiently simple to allow the sequence to be read directly without extensive interpretation. This is in contrast to spectra of underivatized peptides obtained by MALDI/PSD or conventional tandem mass spectrometry, where full sequence interpretation can be challenging. Aided with a set of very simple established rules, it was shown that the sequence of TMPP(+)-Ac derivatives can be derived strictly from predictable fragment ion series. In most cases, this is sufficient to determine extensive, unambiguous, peptide sequences de novo. The partial sequence (35%) of the unknown phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes 130Z was obtained entirely by the mass spectrometric method evaluated here, which provided the basis for evaluating homology and for the design of oligonucleotide probes for cloning the corresponding gene.     

Easy amino acid sequencing of sulfonated peptides using post-source decay on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer equipped with a variable voltage reflector

Tryptic peptides were labeled with sulfonic acid groups at the N-termini using an improved chemistry. The derivatization was performed in common aqueous buffers on peptides adsorbed onto a ZipTip trade mark C(18), thus allowing simultaneous desalting/concentration of the sample. When only Arg-terminating peptides were considered, the procedure from adsorption onto the ZipTip until analysis by MALDI-PSD took about 10 min and several samples could be worked on in parallel. The resulting improved post-source decay (PSD) fragmentation produced spectra containing only y-ions. PSD amino acid sequencing of underivatized and derivatized synthetic peptides was compared. From the sequence information obtained from derivatized peptides isolated by ion selection from tryptic in-gel digests, a protein was correctly identified which was difficult to analyze from an unclear peptide mass fingerprint analysis. The method was also applied to the identification and localization of phosphorylated Ser and Tyr residues in native and synthetic peptides.     

A strategy for rapid and efficient sequencing of Lys-C peptides by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry post-source decay

A modification procedure for Lys-C peptides is described which simplifies the correct assignment of the amino acid sequence. Release of the C-terminal lysine from Lys-C peptides by carboxypeptidase B and subsequent N-terminal acetylation of the resulting peptides leads to predictable shifts of the C- and N-terminal fragment ions in Matrix-assisted laser desorption/ionisation time-of-flight post-source decay mass spectra and facilitates the correct assignment of mostly complete amino acid sequences for oligopeptides. The derived sequences of peptides from unknown proteins were used to search in databases for homologous protein sequences. Our method was applied to an unknown protein isolated from eggs of Drosophila melanogaster, resulting in the identification of a peptidyl prolyl cis-trans-isomerase.     

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.     

Structural analysis of (methyl-esterified) oligogalacturonides using post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The use of post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the structural analysis of ((partly) methyl-esterified) oligogalacturonides (oligoGalA) is described. The fragmentation behavior of purified (un)saturated oligoGalA (degree of polymerization 3-6), methyl-esterified and methyl-glycosydated oligoGalA was studied. General fragmentation patterns are described and used for the elucidation of the positions of methyl esters on partly methyl-esterified oligoGalA. This technique now permits the determination of the position of methyl esters or other substituents on pectic fragments, helping in understanding the mode of action of pectinolytic enzymes.     

Sequencing of peptides phosphorylated on serines and threonines by post-source decay in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In the era of complete genome sequences, biochemical and medical research will focus more on the dynamic proteome of a cell. Regulation of proteins by post-translational modifications, which are not determined by the gene sequence, are already intensively studied. One example is phosphorylation of serines and threonines, probably the single most common cellular regulatory mechanism. In this paper we describe the sequencing of mono- and bisphosphorylated peptides, including identification of the phosphorylation sites, by post-source decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In addition to dephosphorylation of the parent ions, we studied the influence of the phosphate group on the fragmentation of peptides. Generally, peptides phosphorylated on serine and threonine residues displayed no difference in their fragmentation patterns. The intensities of the resulting fragment ion signals depend only on the peptide sequence and not on either the phosphorylated amino acid or its position in the peptide chain. Phosphorylation increased the bond cleavage C-terminal to the phosphorylation site more than 10-fold, resulting in abundant signals, which typically dominated the PSD spectra. The produced C-terminally phosphorylated b-type fragment ions showed characteristic dephosphorylated fragment ions b(n) -H(3)PO(4) (-98 Da) and b(n) -HPO(3) (-80 Da) of higher abundances than the phosphorylated fragment ion. As a second layer to identify the phosphorylation site, all internally phosphorylated fragment ions were accompanied by minor, but always detectable, signals of the dephosphorylated fragment ions. Interpretation of PSD spectra of phosphopeptides was not more complicated than for unphosphorylated peptides, despite the increased number of obtained fragment ion signals.     

Identification and fragmentation study of plasticizers with post-source decay matrix-assisted laser desorption/ionization mass spectrometry

A detailed investigation of the most commonly used plasticizers, such as phthalate, adipate and trimellitate esters, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and post-source decay (PSD) MALDI-TOFMS/MS is described. It is shown that PSD MALDI-TOFMS/MS is capable of unambiguous determination of the different types of plasticizers. The determination of the types of plasticizers from different PVC samples by PSD MALDI-TOFMS/MS, without the need for solvent extraction, is also demonstrated. The fragmentation mechanisms of these plasticizers cationized with protons and sodium ions are also reported.     

Advantages of using nested collision induced dissociation/post-source decay with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: sequencing of novel peptides from wasp venom

We have examined the applicability of the 'nested' collision induced dissociation/post-source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C-terminal fragment ions. Assignment of C-terminal fragment ions enabled calculation of N-terminal fragment masses, leading to differentiation between N-terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply.     

Determination of photomodified oligodeoxynucleotides by exonuclease digestion, matrix-assisted laser desorption/ionization and post-source decay mass spectrometry

A fast method to detect and sequence photomodified oligodeoxynucleotides (ODNs) by exonuclease digestion and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is reported. Upon treatment of modified ODNs with both phosphodiesterase I and phosphodiesterase II, the digestion stops at the sites of photomodification. Post-source decay (PSD) of MALDI-produced ions from two enzymatic digestion end products distinguishes isomers such as 5'-d(T[cis-syn]TAAGC) and 5'-d(CGAAT[cis-syn]T), which have symmetrical or identical compositions at the 3' and 5' ends, respectively. Studies have also been done to follow the kinetics for enzyme degradation of photomodified ODNs. The calculated rate constants from a mathematical treatment of the time-dependent MALDI data clearly show that the enzymatic digestion rate slows as the enzyme approaches the modified site.     

Identification of dinucleoside polyphosphates by matrix-assisted laser desorption/ionisation post-source decay mass spectrometry

Dinucleoside polyphosphates are a group of intra- and extracellular mediators controlling numerous physiological functions. In this study dinucleoside polyphosphates were examined by positive ion matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MADLI-TOFMS). 3-Hydroxypicolinic acid was used as UV-absorbing matrix. For the individual dinucleoside polyphosphates Ap(n)A (n = 2-7), Ap(n)G (n = 2-6) and Gp(n)G (n = 2-6), MALDI post-source decay (PSD) mass spectra were measured. Each mass peak in the MALDI-PSD mass spectra could be assigned to individual fragments of dinucleoside polyphosphates. The comparison of the fragmentation patterns of the dinucleoside polyphosphates presented here demonstrates that dinucleoside polyphosphates preferably cleave to fragment ions consisting of the corresponding mononucleoside polyphosphates as well as the corresponding nucleosides and bases during flight in the field-free drift path of the MALDI mass spectrometer. Therefore, the MALDI-PSD approach described here is suitable for identification of other dinucleoside polyphosphates. The present MALDI-PSD mass spectra may be used as MALDI-PSD mass reference spectra for future identification of dinucleoside polyphosphates and other nucleotides.