Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

High-throughput DNA sequencing has resulted in increasing input in protein sequence databases. Today more than 20 genomes have been sequenced and many more will be completed in the near future, including the largest of them all, the human genome. Presently, sequence databases contain entries for more than 425.000 protein sequences. However, the cellular functions are determined by the set of proteins expressed in the cell--the proteome. Two-dimensional gel electrophoresis, mass spectrometry and bioinformatics have become important tools in correlating the proteome with the genome. The current dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated.     

Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

High-throughput DNA sequencing has resulted in increasing input in protein sequence databases. Today more than 20 genomes have been sequenced and many more will be completed in the near future, including the largest of them all, the human genome. Presently, sequence databases contain entries for more than 425.000 protein sequences. However, the cellular functions are determined by the set of proteins expressed in the cell – the proteome. Two-dimensional gel electrophoresis, mass spectrometry and bioinformatics have become important tools in correlating the proteome with the genome. The current dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated. Received: 16 December 1999 / Accepted: 17 December 1999     

Collision induced decomposition of peptides. Choice of collision parameters

Collision-induced dissociation product ion spectra of a series of doubly charged tryptic peptide ions produced by electrospray ionization were obtained by triple-quadrupole tandem mass spectrometry. The sequence information content of the product ion spectra was explored as a function of collision energy and collision-cell gas pressure for parent ions with molecular masses ranging from 300 to 2000 u. The energy range (at a given pressure) in which the degree of fragmentation is acceptable was found to be narrow for parent ions of a given mass, and the optimal collision energy was observed to exhibit a strong linear correlation with parent ion mass. This observed correlation opens the way for on-line software-controled selection of optimal mass spectrometric conditions in the enzymatic digestion-liquid chromatography-tandem mass spectrometric strategy of amino acid sequencing of proteins.     

Sequence information of peptides and proteins with in-source decay in matrix assisted laser desorption/ionization-time of flight-mass spectrometry

In-source decay coupled with matrix assisted laser desorption/ionization-mass spectrometry, which is a mass spectrometric degradation method for the sequencing of peptides and proteins, has been applied to several different polypeptides and proteins. The influence of the nature of the constituent amino acids on positively charged product ions is described. Relatively small molecular mass peptides produced c-, b-, and/or a-series ions usable for C-terminal sequencing as well as y- and/or z-series ions usable for N-terminal sequencing. The formation of the C-terminal sequencing ions (c, b and a) and the N-terminal sequencing ions (y and z) was strongly dependent on the location(s) of basic arginine and lysine residues. The presence of the arginine and/or lysine residues at the N-terminal region was one-sided in the formation of c-, b-, and/or a-series ions, while the presence of those at the C-terminal region was favorable for the formation of y- and z-series ions. In-source decay experiments of intact proteins, apomyoglobin and two viral coat proteins, led to large amounts of c-series ions and small amounts of y-series ions, which reflected internal sequences.     

Bioactive peptides from the skin of ranid frogs: modern approaches to the mass spectrometric <Emphasis Type="Italic">de novo</Emphasis> sequencing

A mass spectrometric approach to the de novo sequencing of peptides containing up to 50 amino acid residues was proposed. Arising problems of the mass spectrometric sequencing and their solution were considered on specific examples. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1061–1072, May, 2008.     

Use of nonspecific cleavage products for protein sequence analysis as shown on calcyclin isolated from human granulocytes

In this paper, analysis strategies developed for a sequencing problem concerning the identification of an S100 protein isolated from human granulocytes are discussed. The analysis of a trypsinized lyophilized sample suggested the presence of a number of peptides which are non-tryptic in origin. During purification of proteins from cell lysates nonspecific cleavage can be observed which may reflect biological processes and can become an unavoidable analytical problem. Current mass spectrometric software is evaluated for the analysis of nonspecific digests in this context. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), high-performance liquid chromatography (HPLC)-MS/MS, and selected ion monitoring (SIM)-MS/MS have been used for peptide analysis and in addition HPLC-MS was carried out for protein analysis leading to the detection of an N-terminal modification of the protein. The success of the study is mainly due to the careful investigation of nonspecific cleavage products. Data obtained from the routine mass spectrometric analysis of an in-gel-digest allowed the identification of this protein as S100 calcium-binding protein A6-calcyclin whose expression in granulocytes has not been described so far.     

C-Terminal sequencing of protein by MALDI mass spectrometry through the specific derivatization of the α-carboxyl group with 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide

We present here an approach to C-terminal sequencing of proteins by the procedure consisting of the following: (1) derivatization of the C-terminal α-carboxyl group with 3-aminopropyltris(2,4,6-trimethoxyphenyl)-phosphonium bromide (TMPP-propylamine) through oxazolone chemistry, (2) enzymatic proteolysis of the TMPP-derivatized protein, and (3) MALDI-MS/MS analysis of the peptide mixture, in which the C-terminal peptide incorporating the TMPP group is preferably detected. In this protocol, it is possible to choose any endoproteinase such as trypsin, GluC, and AspN for digestion so that a C-terminal peptide with length appropriate for mass spectrometric sequencing could be generated. The peptide labeled with TMPP-propylamine at the C terminus tends to exhibit y-type ions in MS/MS spectra, allowing manual sequence interpretation with the simplified fragmentation pattern. The efficacy of the method was verified with five proteins, which demonstrated that the C-terminal peptides were readily distinguishable by their peak intensity and characteristic mass signature peak in MALDI-PSD analysis.     

Mass spectrometric approach for the analysis of food proteins

In the study of food proteins, the need for accurate protein structural analysis has been acknowledged because of the fact that nucleotide sequencing alone is of limited analytical value if not combined with relevant information regarding the specific protein expressed and the occurrence of phosphorylation, glycosylation and disulphide bridges, and with the modification induced by the technological treatment. Mass spectrometry, whether used alone or to complement the traditional molecular-based techniques has become fundamental to the structural analysis of proteins. It is, moreover, virtually irreplaceable in determining post-translational modifications as conventional methods cannot deliver reliable data. What lies at the root of this methodological breakthrough is the combination of high-resolution separation techniques such as two-dimensional electrophoresis or capillary reverse- phase high-performance liquid chromatography with mass spectrometric analysis, what is termed "proteomic" analysis. Thus, it appears appropriate to state that the new mass spectrometric techniques have been established as a valuable and efficient tool for protein and peptide analysis in complex mixtures, like those from food matrices, enabling us therefore to provide accurate information on molecular weight and also to put forth a structural assessment at a low-picomole level of material. Thus, a series of alternative approaches have been developed based on advanced mass spectrometric analysis in conjunction with classic protein chemistry in order to provide an in-depth view of food protein structure. This review outlines several of these novel methodologies as they apply to structural characterization of food products.     

Applications of mass spectrometry to food proteins and peptides

The application of mass spectrometry (MS) to large biomolecules has been revolutionized in the past decade with the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) techniques. ESI and MALDI permit solvent evaporation and sublimation of large biomolecules into the gaseous phase, respectively. The coupling of ESI or MALDI to an appropriate mass spectrometer has allowed the determination of accurate molecular mass and the detection of chemical modification at high sensitivity (picomole to femtomole). The interface of mass spectrometry hardware with computers and new extended mass spectrometric methods has resulted in the use of MS for protein sequencing, post-translational modifications, protein conformations (native, denatured, folding intermediates), protein folding/unfolding, and protein-protein or protein-ligand interactions. In this review, applications of MS, particularly ESI-MS and MALDI time-of-flight MS, to food proteins and peptides are described.     

Electrospray-assisted laser desorption/ionization and tandem mass spectrometry of peptides and proteins

We have constructed an electrospray-assisted laser desorption/ionization (ELDI) source which utilizes a nitrogen laser pulse to desorb intact molecules from matrix-containing sample solution droplets, followed by electrospray ionization (ESI) post-ionization. The ELDI source is coupled to a quadrupole ion trap mass spectrometer and allows sampling under ambient conditions. Preliminary data showed that ELDI produces ESI-like multiply charged peptides and proteins up to 29 kDa carbonic anhydrase and 66 kDa bovine albumin from single-protein solutions, as well as from complex digest mixtures. The generated multiply charged polypeptides enable efficient tandem mass spectrometric (MS/MS)-based peptide sequencing. ELDI-MS/MS of protein digests and small intact proteins was performed both by collisionally activated dissociation (CAD) and by nozzle-skimmer dissociation (NSD). ELDI-MS/MS may be a useful tool for protein sequencing analysis and top-down proteomics study, and may complement matrix-assisted laser desorption/ionization (MALDI)-based measurements.     

A proteomic analysis of organelles from Arabidopsis thaliana

We introduce the use of Arabidopsis thaliana callus culture as a system for proteomic analysis of plant organelles using liquid-grown callus. This callus is relatively homogeneous, reproducible and cytoplasmically rich, and provides organelles in sufficient quantities for proteomic studies. A database was generated of mitochondrial, endoplasmic reticulum (ER), Golgi/prevacuolar compartment and plasma membrane (PM) markers using two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) and peptide sequencing or mass spectrometric methods. The major callus membrane-associated proteins were characterised as being integral or peripheral by Triton X-114 phase partitioning. The database was used to define specific proteins at the Arabidopsis callus plasma membrane. This database of organelle proteins provides the basis for future characterisation of the expression and localisation of novel plant proteins.     

Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry

Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein.     

Sequential electroelution and mass spectroscopic identification of intact sodium dodecyl sulfate-proteins labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester

A gel electrophoresis apparatus capable of scanning the migration path fluorometrically and of computer-directed electroelution of bands was applied to the mass spectrometric identification of sequentially electroeluted 5(6)-carboxyfluorescein-N-hydrosuccinimide ester (FLUOS)-labeled sodium dodedyl sulfate (SDS)-proteins. The masses of four electroeluted SDS-proteins under study determined by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry are changed by 1% due to their reaction with FLUOS in a 1:5 molar ratio of protein:label, allowing for the identification of the labeled intact proteins on the basis of mass. More importantly, the partial (10 or 50%) derivatization of proteins with FLUOS does not preclude their tryptic hydrolysis, and identification of the protein on the basis of the mass spectrometric analysis of its tryptic peptides. Potentially, the procedure allows for the automated mass spectrometric identification of SDS-proteins globally labeled with FLUOS and electrophoretically separated, without need for any gel sectioning.     

Accurate mass as a bioinformatic parameter in data-to-knowledge conversion: Fourier transform ion cyclotron resonance mass spectrometry for peptide de novo sequencing

With the availability of ultra-precise mass spectrometric biomolecular data, the accurate mass is becoming a physical quantity of high interest for bioinformatics tools and strategies. Fourier transform ion cyclotron resonance mass spectrometry with electrospray ionization or matrix-assisted laser desorption/ionization sources now allows the easy determination of amino acid composition of medium size, unknown peptides when employing combinatorial calculation of hypothetical parent and fragment ion masses. This new method, which in a second step, allows the reliable de-novo sequencing of completely unknown peptides ["composition-based sequencing (CBS)"(1)] appears to open a wide new field of bioanalytical investigation. It has been shown that even unspecifically cleaved proteins can be identified easily and reliably when accurate mass evaluation is combined with protein database search tools.(2) It is quite clear that, while the nominal mass of a peptide has obviously no useful correlation to biomolecular structure, the accurate mass, instead, has a strong and direct correlation to structure that so far has not been exploited considerably by bioinformatic tools. It has already become obvious that accurate mass evaluation is going to become a central goal for bioinformatics strategies in the near future.(3-11) Strategies for extracting structural, and even functional, information out of accurate mass values of biomolecules still have to be developed. Examples and prospects of accurate mass evaluation in bioinformatics for the field of proteomics are outlined in the following.     

Isotope-coded N-terminal sulfonation of peptides allows quantitative proteomic analysis with increased de novo peptide sequencing capability

Recently various methods for the N-terminal sulfonation of peptides have been developed for the mass spectrometric analyses of proteomic samples to facilitate de novo sequencing of the peptides produced. This paper describes the isotope-coded N-terminal sulfonation (ICenS) of peptides; this procedure allows both de novo peptide sequencing and quantitative proteomics to be studied simultaneously. As N-terminal sulfonation reagents, 13C-labeled 4-sulfophenyl[13C6]isothiocyanate (13C-SPITC) and unlabeled 4-sulfophenyl isothiocyanate (12C-SPITC) were synthesized. The experimental and reference peptide mixtures were derivatized independently using 13C-SPITC and 12C-SPITC and then combined to generate an isotopically labeled peptide mixture in which each isotopic pair differs in mass by 6 Da. Capillary reverse-phase liquid chromatography/tandem mass spectrometry experiments on the resulting peptide mixtures revealed several immediate advantages of ICenS in addition to the de novo sequencing capability of N-terminal sulfonation, namely, differentiation between N-terminal sulfonated peptides and unmodified peptides in mass spectra, differentiation between N- and C-terminal fragments in tandem mass spectra of multiply protonated peptides by comparing fragmentations of the isotopic pairs, and relative peptide quantification between proteome samples. We demonstrate that the combination of N-terminal sulfonation and isotope coding in the mass spectrometric analysis of proteomic samples is a viable method that overcomes many problems associated with current N-terminal sulfonation methods.     

Characterization of the P13 membrane protein of Borrelia burgdorferi by mass spectrometry

Borrelia burgdorferi sensu lato is a tick-borne pathogen that causes Lyme disease. The characterization of membrane proteins from this and other pathogens may yield a better understanding of the mechanisms of infection and information useful for vaccine design. Characterization of the highly hydrophobic Borrelia outer membrane component P13 from a mutant (OspA- OspB- OspC- and OspD-) strain was undertaken by use of a combination of mass spectrometric methods. In a previous investigation, an electrospray ionization (ESI) mass spectrum of the intact protein provided an average molecular weight that was 20 Da lower than the predicted molecular weight. The mass deviation could be explained by a modification of the N-terminus of the protein such as pyroglutamylation (-17 Da) in combination with the experimental error of measurement, however more information was required. New structural information for this membrane protein was provided by peptide mapping with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) and sequencing with ESI-quadrupole-TOF tandem MS.     

Characterization of proteins by mass spectrometry. Invited lecture.

Plasma desorption and fast atom bombardment mass spectrometry have in the last decade demonstrated the potential of mass spectrometry for protein studies. The recently developed matrix-assisted laser desorption and electrospray mass spectrometry have expanded the analytical potential of mass spectrometry to cover nearly all proteins. The type of information obtained with the four methods is described and their performances are compared. The potential of combining mass spectrometric relative molecular mass information on proteins with the information contained in protein sequence databases is outlined and some typical fields of application of mass spectrometry in protein chemistry are described. The need for the full integration of mass spectrometry in the protein laboratory is discussed.     

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.     

Tumor pleural effusion proteome profiling for ovarian cancer biomarkers mining

Mass spectrometric proteome profiling of tumor pleural effusion (TPE) liquid fraction from ovarian cancer patients was performed to identify the potential biomarkers of the disease. The methodology of analysis of the TPE protein composition included the removal of high-abundant proteins by affinity chromatography, additional fractionation of the low-abundant proteins based on their lipophilicity, and high-resolution mass spectrometric analysis. As a result, 190 proteins were indentified, 49% of them belonging to the groups of extracellular and membrane proteins. The application of several criteria to data analysis allowed us to generate a group of 26 proteins that are promising candidates for testing as ovarian cancer biomarkers.     

Current approaches for global post-translational modification discovery and mass spectrometric analysis

More and more attention is being focused on the analysis of post-translational modifications (PTMs) on proteins as researchers are continually learning how essential they are for proper cellular function. As there are hundreds of different types of known PTMs, traditional methods of modification analysis are incapable of comprehensively monitoring for post-translational modifications, a task which is a necessity for truly understanding a cell's biology. This review highlights recent developments in novel multiplexed methods of PTM analysis including: fluorescent stain and immuno-based methods, hardware-based mass spectrometric methods and computational-based mass spectrometric methods. Many of these techniques show great promise and will likely be a valuable resource for the biological community.