Proteomic analysis of carbonylated proteins in two-dimensional gel electrophoresis using avidin-fluorescein affinity staining

A method for detecting carbonylated proteins in two-dimensional electrophoresis (2-DE) was developed using biotinylation and avidin-fluorescein isothiocyanate (FITC) affinity staining. The method was used to examine oxidatively modified proteins associated with oxidative stress. Carbonyl formation in proteins was first examined in a model system by subjecting bovine serum albumin (BSA) and ribonuclease A (RNase A) to metal-catalyzed oxidation (MCO). Carbonyl group formation was found to occur at multiple sites along with a small amount of polypeptide chain cleavage. In vivo studies were conducted in yeast cell cultures using 5 mM hydrogen peroxide to induce oxidative stress. Biotinylation of yeast protein was accomplished during extraction at 4 degrees C in a lysis buffer containing 5 mM biotin-hydrazide. Biotin-hydrazide forms a Schiff base with a carbonyl group on an oxidized protein that is subsequently reduced before electrophoresis. Proteins were separated by either 2-DE or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biotinylated species were detected using avidin-FITC affinity staining. Detection sensitivity with biotinylated proteins was five times higher than achieved by silver staining. The limit of detection with avidin-FITC staining approached 0.64 pmol of protein-associated carbonyls. Twenty carbonylated proteins were identified in the proteome of yeast following oxidative stress with hydrogen peroxide. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis of tryptic peptides was used to identify peptides extracted from gels. Aconitase, heat shock protein SSA1 and SSC1, pyruvate decarboxylase isozyme 1, pyruvate kinase 1, enolase 1 and 2, phosphoglycerate kinase, fructose-bisphosphate aldorase, and glyceraldehyde-3-phosphate dehydrogenase were among the major targets of oxidative stress.     

Identification of yeast oxidized proteins: chromatographic top-down approach for identification of carbonylated, fragmented and cross-linked proteins in yeast

The effects of oxidative stress on the yeast proteome were studied using hydrogen peroxide as the stress agent. Oxidized proteins were isolated by (1) biotinylation of oxidized proteins with biotin hydrazide, (2) affinity selection using monomeric avidin affinity chromatography, and (3) further fractionated by reversed-phase liquid chromatography (RPLC) on a C(8) column. Oxidized protein fractions from RPLC were then trypsin digested and the peptide cleavage fragments identified by tandem mass spectrometry (MS/MS). Slightly over 400 proteins were identified. Sites of carbonyl formation were found in roughly one fourth of these proteins. Oxidation on other amino acids in carbonylated peptides was seen in 32 cases while carbonylation was absent in 96 of the oxidized proteins observed. Although there are large numbers of potential oxidation sites, oxidation seemed to be restricted to a small area in most of the proteins identified. Sometimes multiple amino acids in the same tryptic peptide were oxidized. A second trend was that more than 8% of the proteins identified appeared in more than one of the RPLC fractions. Based on the position of the peptides identified in the primary structure of protein candidates derived from databases it was concluded that this occurred by fragmentation of a parent protein. It is not clear from the data whether the fragmentation process was of enzymatic or oxidative origin. Finally, peptides from two or more proteins occurred together in more than one reversed phase fraction with 2% of the proteins identified. This data was interpreted to mean that this was the result of protein cross-linking.     

Dansyl labeling and bidimensional mass spectrometry to investigate protein carbonylation

Carbonylation is a non-enzymatic irreversible post-translational modification. The adduction of carbonyl groups to proteins is due to the presence of excess of ROS in cells. Carbonylation of specific amino acid side chains is one of the most abundant consequences of oxidative stress; therefore, the determination of carbonyl groups content in proteins is regarded as a reliable way to estimate the cellular damage caused by oxidative stress. This paper reports a novel RIGhT (Reporter Ion Generating Tag) (A. Amoresano, G. Monti, C. Cirulli, G. Marino. Rapid Commun. Mass Spectrom. 2006, 20, 1400) approach for selective labeling of carbonyl groups in proteins using dansylhydrazide, coupled with selective analysis by bidimensional mass spectrometry. We first applied this approach to ribonuclease A and lysozyme as model proteins. According to the so-called 'gel-free procedures', the analysis is carried out at the level of peptides following tryptic digest of the whole protein mixture. Modified RNaseA was analyzed in combined MS(2) and MS(3) scan mode, to specifically select the dansylated species taking advantage of the dansyl-specific fragmentation pathways. This combination allowed us to obtain a significant increase in signal/noise ratio and a significant increase in sensitivity of analysis, due to the reduction of duty cycle of the mass spectrometer. The unique signal obtained was correlated to peptide 1-10 of RNaseA carbonylated and labeled by dansylhydrazide. This strategy represents the first method leading to the direct identification of the carbonylation sites in proteins, thus indicating the feasibility of this strategy to investigate protein carbonylation in a proteomic approach.     

Isotope-coded dimethyl tagging for differential quantification of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-product of lipid peroxidation

Peroxidation of cellular membrane lipids, rich in polyunsaturated fatty acids, generates electrophilic, α, β-unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE). HNE is a highly reactive and cytotoxic molecule that can react with the nucleophilic sites in proteins causing posttranslational modification. The identification of protein targets is an important first step; however, quantitative profiling of site-specific modifications is necessary to understand the biological impact of HNE-induced carbonylation. We report a method that uses light (H(12)CHO) and heavy (D(13)CDO) isotopic variant of formaldehyde to differentially label primary amines (N-termini and ε-amino group of lysines) in peptides through reductive methylation and, combined with selective enrichment of modified peptides, permits comparison of the extent of carbonylation in two samples after mixing for simultaneous liquid chromatography-mass spectrometry. Specifically, dimethyl-labeled peptide carbonyls were fractionated from unmodified peptides using solid-phase hydrazide chemistry to immobilize them to porous glass beads and, after removing the unmodified peptides by thoroughly washing the beads, subsequently recover them by acid-catalyzed hydrolysis. The method was developed using HNE-modified synthetic peptides and also showing enrichment from a complex matrix of digested human plasma proteins. Applicability was confirmed using apomyoglobin as an analyte, implicating thereby its potential value to proteome-wide identification and relative quantification of posttranslational protein carbonylation with residue-specific information. Because HNE attachment may not necessarily cause change in protein abundance, this modification-focused quantification should facilitate the characterization of accompanied changes in protein function and, also, provide important insights into molecular signaling mechanisms and a better understanding of cellular processes associated with oxidative stress.     

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.     

Characterization of metal-labelled peptides by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry

Metal labelling of peptides and proteins using high-affinity metal-chelating compounds has found widespread applications in the medical and bioanalytical fields. In the present study we investigated the analysis of peptides derivatized either with cysteine- or amino group-directed metal-bound DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The metal complexes of DOTA were shown to be stable under MALDI-MS conditions. The introduction of the metal label led in a number of cases to significantly increased signal-to-noise (S/N) values and thus improved sensitivity of the labelled peptides compared to their unlabelled counterparts, especially for multiply labelled peptides. The presence of the labels did alter the tandem mass spectrometric (MS/MS) behaviour, namely the formation of sequence specific a-, b- and y-ion series, in dependence of the position of the label within the peptide sequence. For cysteine-derivatized peptides several label-specific reporter ions and characteristic immonium ions could be identified. Amino-directed labelling led only to the formation of characteristic immonium ions in ε-amino groups of lysine, whereas N-terminal labelling in some cases led to the formation of a(1)- and b(1)-ions. The results clearly show that MALDI-MS is suitable for the analysis of metal-labelled peptides, which was also confirmed in liquid chromatography (LC)/MALDI-based identification of proteins in a model protein mixture labelled with Cys-reactive DOTA. Here, in comparison to a run with alkylated cysteines, more than 50% more cysteine-containing peptides were identified.     

Strategy for identification and detection of multiple oxidative modifications within proteins applied on persulfate-oxidized hemoglobin and human serum albumin

Oxidative stress has been suggested as an underlying mechanism of many human diseases. However, definitive evidence for this association has not been presented due to different shortcomings of the methods used to measure biomarkers of oxidative stress. Persulfates are oxidizing agents known to elicit hypersensitive reactions from the airways and skin. Despite a frequent use of persulfates at many work places, no biomarkers for persulfate exposure are available. The aim of this study was to develop a strategy for the identification and detection of multiple oxidative modifications within proteins. This strategy was applied on persulfate-oxidized proteins to identify oxidized peptides suitable for further investigation as biomarkers of persulfate exposure or oxidative stress. A strategy for the identification and the relative quantification of multiple oxidative modifications within proteins was developed. The usage of two software packages facilitated the search for modified peptides to a great extent. Oxidized peptides were relatively quantified using liquid chromatography/tandem mass spectrometry in selected reaction monitoring mode. The result showed that persulfates oxidize tryptophans and methionines resulting in mass shifts of 16 and/or 32 Da. Also, oxidized albumin peptides in nasal lavage fluid samples from subjects challenged with persulfate were detected. The oxidation degree before and after challenge remained constant for peptides containing methionine sulfoxide. For peptides containing oxidized tryptophan the oxidation degree increased after exposure. Some of these oxidized peptides may be suitable as biomarkers; however, further evaluation is required.     

Applications of Tandem Mass Spectrometry (MS/MS) in Protein Analysis for Biomedical Research

Mass Spectrometry (MS) allows the analysis of proteins and peptides through a variety of methods, such as Electrospray Ionization-Mass Spectrometry (ESI-MS) or Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). These methods allow identification of the mass of a protein or a peptide as intact molecules or the identification of a protein through peptide-mass fingerprinting generated upon enzymatic digestion. Tandem mass spectrometry (MS/MS) allows the fragmentation of proteins and peptides to determine the amino acid sequence of proteins (top-down and middle-down proteomics) and peptides (bottom-up proteomics). Furthermore, tandem mass spectrometry also allows the identification of post-translational modifications (PTMs) of proteins and peptides. Here, we discuss the application of MS/MS in biomedical research, indicating specific examples for the identification of proteins or peptides and their PTMs as relevant biomarkers for diagnostic and therapy.     

Novel biomarkers of protein oxidation sites and degrees using horse cytochrome c as the target by mass spectrometry

Biomarkers held both incredible application and significant challenge in probing the oxidation mechanisms of proteins under oxidative stress. Here, mass spectrometry (MS) coupled with liquid chromatography (LC) was applied to establish a new pipeline to probe the oxidation sites and degrees of horse cytochrome c (HCC) with its oxidative products serving as the biomarkers. Samples of native and UV/H(2)O(2) oxidized HCCs were digested by trypsin and subjected to biomarker discovery using LC/MS and tandem mass spectrometry (MS/MS). Experiment results proved that the main oxidation sites were located at Cys(14), Cys(17), Met(65) and Met(80) residues in peptides C(14)AQC(heme)HTVEK(22), C(14)AQCHTVEK(22), E(60)ETLMEYLENPKK(73), M(80)IFAGIK(86) and M(80)IFAGIKK(87). Quantitative analysis on the oxidized peptides showed the oxidation degrees of target sites had positive correlations with extended oxidation dose and controlled by residues types and their accessibility to solvent molecules. Being able to provide plentiful information for the oxidation sites and oxidation degrees, the identified oxidized products were feasibility biomarkers for HCC oxidation, compared with the conventional protein carbonyl assay.     

Analysis of Biomolecules by Atmospheric Pressure Visible-Wavelength MALDI-Ion Trap-MS in Transmission Geometry

We report the development of a new AP visible-wavelength MALDI-ion trap-MS instrument with significantly improved performance over our previously reported system (Int. J. Mass Spectrom. 315, 66–73 (2012)). A Nd:YAG pulsed laser emitting light at 532 nm was used to desorb and ionize oligosaccharides and peptides in transmission geometry through a glass slide. Limits of detection (LODs) achieved in MS mode correspond to picomole quantities of oligosaccharides and femtomole quantities of peptides. Tandem MS (MS/MS) experiments enabled identification of enzymatically digested proteins and oligosaccharides by comparison of MS/MS spectra with data found in protein and glycan databases. Moreover, the softness of ionization, LODs, and fragmentation spectra of biomolecules by AP visible-wavelength MALDI-MS were compared to those obtained by AP UV MALDI-MS using a Nd:YAG laser emitting light at 355 nm. AP visible-wavelength MALDI appears to be a softer ionization technique then AP UV MALDI for the analysis of sulfated peptides, while visible-wavelength MALDI-MS, MS/MS, and MS/MS/MS spectra of other biomolecules analyzed were mostly similar to those obtained by AP UV MALDI-MS. Therefore, the methodology presented will be useful for MS and MSⁿ analyses of biomolecules at atmospheric pressure. Additionally, the AP visible-wavelength MALDI developed can be readily used for soft ionization of analytes on various mass spectrometers.

In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics

We present a new isotopic labeling strategy to modify the N-terminal amino group of peptides in a quantifiable reaction without the use of expensive reagents or solvents. The In Vacuo Isotope Coded Alkylation Technique (IVICAT) is a methylation reaction, carried out at low pressure (<100 mTorr), that results in a stable quaternary trimethylammonium group, thus adding a permanent positive charge at the N-terminus of peptides without modifying the epsilon-amino groups of lysine. The methylation reaction increases the signal intensity of modified peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography (LC)/MS and the isotopic peak pair differs by 9 mass units which can be easily resolved by either instrument. N-terminally trimethylated peptides exhibit collision-induced dissociation (CID) mass spectra that differ from their unmodified analogues by an enhanced b-ion series in MS2 spectra due to the fixed positive charge. Using LC/MS/MS with an LTQ mass analyzer for quantification, the experimentally determined ratios of H9- to D9-trimethyl-labeled peptides of beta-casein provided accurate estimates of the actual ratios with low % error. IVICAT labeling also accurately quantified proteins in rat kidney inner medullary collecting duct cell types, as judged by comparison with relative quantification by subsequent immunoblotting experiments. IVICAT labeling, when used in conjunction with the new proteomics software QUIL, can accurately report relative protein abundances and increase the sequence coverage of proteins of tissue proteomes.     

Microscopic analysis of protein oxidative damage: effect of carbonylation on structure, dynamics, and aggregability of villin headpiece

One of the most important irreversible oxidative modifications of proteins is carbonylation, the process of introducing a carbonyl group in reaction with reactive oxygen species. Notably, carbonylation increases with the age of cells and is associated with the formation of intracellular protein aggregates and the pathogenesis of age-related disorders such as neurodegenerative diseases and cancer. However, it is still largely unclear how carbonylation affects protein structure, dynamics, and aggregability at the atomic level. Here, we use classical molecular dynamics simulations to study structure and dynamics of the carbonylated headpiece domain of villin, a key actin-organizing protein. We perform an exhaustive set of molecular dynamics simulations of a native villin headpiece together with every possible combination of carbonylated versions of its seven lysine, arginine, and proline residues, quantitatively the most important carbonylable amino acids. Surprisingly, our results suggest that high levels of carbonylation, far above those associated with cell death in vivo, may be required to destabilize and unfold protein structure through the disruption of specific stabilizing elements, such as salt bridges or proline kinks, or tampering with the hydrophobic effect. On the other hand, by using thermodynamic integration and molecular hydrophobicity potential approaches, we quantitatively show that carbonylation of hydrophilic lysine and arginine residues is equivalent to introducing hydrophobic, charge-neutral mutations in their place, and, by comparison with experimental results, we demonstrate that this by itself significantly increases the intrinsic aggregation propensity of both structured, native proteins and their unfolded states. Finally, our results provide a foundation for a novel experimental strategy to study the effects of carbonylation on protein structure, dynamics, and aggregability using site-directed mutagenesis.     

Applications of MALDI-MS/MS-Based Proteomics in Biomedical Research

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is one of the most widely used techniques in proteomics to achieve structural identification and characterization of proteins and peptides, including their variety of proteoforms due to post-translational modifications (PTMs) or protein–protein interactions (PPIs). MALDI-MS and MALDI tandem mass spectrometry (MS/MS) have been developed as analytical techniques to study small and large molecules, offering picomole to femtomole sensitivity and enabling the direct analysis of biological samples, such as biofluids, solid tissues, tissue/cell homogenates, and cell culture lysates, with a minimized procedure of sample preparation. In the last decades, structural identification of peptides and proteins achieved by MALDI-MS/MS helped researchers and clinicians to decipher molecular function, biological process, cellular component, and related pathways of the gene products as well as their involvement in pathogenesis of diseases. In this review, we highlight the applications of MALDI ionization source and tandem approaches for MS for analyzing biomedical relevant peptides and proteins. Furthermore, one of the most relevant applications of MALDI-MS/MS is to provide “molecular pictures”, which offer in situ information about molecular weight proteins without labeling of potential targets. Histology-directed MALDI-mass spectrometry imaging (MSI) uses MALDI-ToF/ToF or other MALDI tandem mass spectrometers for accurate sequence analysis of peptide biomarkers and biological active compounds directly in tissues, to assure complementary and essential spatial data compared with those obtained by LC-ESI-MS/MS technique.     

Use of an integrated MS--multiplexed MS/MS data acquisition strategy for high-coverage peptide mapping studies

Liquid chromatography/mass spectrometry (LC/MS) peptide maps have become a basic tool for characterizing proteins of biological and pharmaceutical interest. The ability to generate reproducible maps with high protein sequence coverage is a central goal of methods development. We have applied a recently developed analytical approach (termed LC/MS(E)) to LC/MS peptide mapping. Using the LC/MS(E) approach, the mass detector alternates between a low-energy scanning mode (MS) for accurate mass peptide precursor identification, and an elevated-energy mode (MS(E)) for generation of accurate mass multiplex peptide fragmentation data. In this paper, we evaluate this analytical approach against a tryptic digest of yeast enolase. From the low-energy data, high peptide map coverage (98% of sequence from peptides >3 amino acids) was reproducibly obtained. The MS signal for essentially equimolar peptides varied over 2 orders of magnitude in intensity, and peptide intensities could be precisely and reproducibly measured. Using the temporal constraint that MS(E) peptide fragment ions exhibit chromatographic profiles that parallel the precursor ions that generated them, we were able to produce accurate mass time-resolved MS/MS information for all enolase peptides with sufficient abundance to produce a detectable fragment ion.     

Liquid chromatography/tandem mass spectrometry characterization of oxidized amyloid beta peptides as potential biomarkers of Alzheimer's disease

Alzheimer's disease is characterized by the deposition of senile plaques that consist primarily of amyloid beta peptides. There is substantial evidence that amyloid beta is oxidized in vivo, which has led to the suggestion that oxidative stress is an important mediator of Alzheimer's disease. Metal-catalyzed oxidation can mimic in vivo oxidation of amyloid beta because the metal ion binds to the amino acid residues at the site of oxidation, which then deliver reactive oxygen species to that site. Based on electrospray mass spectrometry, it has been suggested that metal-catalyzed oxidation occurs on histidines-13 and -14. Unfortunately, the amyloid beta peptides provide complex spectra, so it is difficult to definitively characterize the sites of oxidation. Trypsin digestion of both native and oxidized amyloid beta1-16 and amyloid beta1-40 resulted in the formation of tryptic peptides corresponding to amyloid beta6-16, which could be separated by liquid chromatography (LC). Sites of oxidation were then unequivocally characterized as histidine-13 and histidine-14 by LC/tandem mass spectrometric (MS/MS) analysis of the tryptic peptides. The ability to analyze the specific amyloid beta6-16 tryptic fragments derived from full-length amyloid beta peptides will make it possible to determine whether oxidation in vivo occurs at specific histidine residues and/or at other amino acid residues such as methionine-35. Using methodology based on LC/MS/MS it will also be possible to analyze the relative amounts of oxidized peptides and native peptide in cerebrospinal fluid from patients with Alzheimer's disease as biomarkers of oxidative stress.     

Site-specific N-glycosylation analysis: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides

The identification of glycosylation sites in proteins is often possible through a combination of proteolytic digestion, separation, mass spectrometry (MS) and tandem MS (MS/MS). Liquid chromatography (LC) in combination with MS/MS has been a reliable method for detecting glycopeptides in digestion mixtures, and for assigning glycosylation sites and glycopeptide sequences. Direct interfacing of LC with MS relies on electrospray ionization, which produces ions with two, three or four charges for most proteolytic peptides and glycopeptides. MS/MS spectra of such glycopeptide ions often lead to ambiguous interpretation if deconvolution to the singly charged level is not used. In contrast, the matrix-assisted laser desorption/ionization (MALDI) technique usually produces singly charged peptide and glycopeptide ions. These ions require an extended m/z range, as provided by the quadrupole-quadrupole time-of-flight (QqTOF) instrument used in these experiments, but the main advantages of studying singly charged ions are the simplicity and consistency of the MS/MS spectra. A first aim of the present study is to develop methods to recognize and use glycopeptide [M+H]+ ions as precursors for MS/MS, and thus for glycopeptide/glycoprotein identification as part of wider proteomics studies. Secondly, this article aims at demonstrating the usefulness of MALDI-MS/MS spectra of N-glycopeptides. Mixtures of diverse types of proteins, obtained commercially, were prepared and subjected to reduction, alkylation and tryptic digestion. Micro-column reversed-phase separation allowed deposition of several fractions on MALDI plates, followed by MS and MS/MS analysis of all peptides. Glycopeptide fractions were identified after MS by their specific m/z spacing patterns (162, 203, 291 u) between glycoforms, and then analyzed by MS/MS. In most cases, MS/MS spectra of [M+H]+ ions of glycopeptides featured peaks useful for determining sugar composition, peptide sequence, and thus probable glycosylation site. Peptide-related product ions could be used in database search procedures and allowed the identification of the glycoproteins.     

Disposable chromatography for a high-throughput nano-ESI/MS and nano-ESI/MS-MS platform

High-throughput proteomics has typically relied on protein identification based on MALDI-MS peptide maps of proteolytic digests of 2D-gel-separated proteins. This technique, however, requires significant sequence coverage in order to achieve a high level of confidence in the identification. Tandem MS data have the advantage of requiring fewer peptides (2) for high confidence identification, assuming adequate MS/MS sequence coverage. MALDI-MS/MS techniques are becoming available, but can still be problematic because of the difficulty of inducing fragment ions of a singly charged parent ion. Electrospray ionization, however, has the advantage of generating multiply charged species that are more readily fragmented during MS/MS analysis. Two electrospray/tandem mass spectrometry-based approaches, nanovial-ESI-MS/MS and LC-MS/MS, are used for high throughput proteomics, but much less often than MALDI-MS and peptide mass fingerprinting. Nanovial introduction entails extensive manual manipulation and often shows significant chemical background from the in-gel digest. LC-MS has the advantages that the chemical background can be removed prior to analysis and the analytes are concentrated during the separation, resulting in more abundant analyte signals. On the other hand, LC-MS can often be time intensive. Here, we report the incorporation of on-line sample clean-up and analyte concentration with a high-throughput, chip-based, robotic nano-ESI-MS platform for proteomics studies.     

Mass spectrometric characterization of peptides containing different oxidized tryptophan residues

The term reactive oxygen species refers to small molecules that can oxidize, for example, nearby proteins, especially cysteine, methionine, tryptophan, and tyrosine residues. Tryptophan oxidation is always irreversible in the cell and can yield several oxidation products, such as 5-hydroxy-tryptophan (5-HTP), oxindolylalanine (Oia), kynurenine (Kyn), and N-formyl-kynurenine (NFK). Because of the severe effects that oxidized tryptophan residues can have on proteins, there is a great need to develop generally applicable and highly sensitive techniques to identify the oxidized residue and the oxidation product. Here, the fragmentation behavior of synthetic peptides corresponding to sequences recently identified in three skeletal muscle proteins as containing oxidized tryptophan residues were studied using postsource decay and collision-induced dissociation (CID) in matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry (MS) and CID in an electrospray ionization (ESI) double quadrupole TOF-MS. For each sequence, a panel of five different peptides containing Trp, 5-HTP, Kyn, NFK, or Oia residue was studied. It was always possible to identify the modified positions by the y-series and also to distinguish the different oxidation products by characteristic fragment ions in the lower mass range by tandem MS. NFK- and Kyn-containing peptides displayed an intense signal at m/z 174.1, which could be useful in identifying accordingly modified peptides by a sensitive precursor ion scan. Most importantly, it was always possible to distinguish isomeric 5-HTP and Oia residues. In ESI- and MALDI-MS/MS, this was achieved by the signal intensity ratios of two signals obtained at m/z 130.1 and 146.1. In addition, high collision energy CID in the MALDI-TOF/TOF-MS also permitted the identification of these two isomeric residues by their v- and w-ions, respectively.     

Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis

Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis.     

Proteome profile of the MCF7 cancer cell line: a mass spectrometric evaluation

The development of novel proteomic technologies that will enable the discovery of disease specific biomarkers is essential in the clinical setting to facilitate early diagnosis and increase survivability rates. We are reporting a shotgun two-dimensional (2D) strong cationic exchange/reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (SCX/RPLC/ESI-MS/MS) protocol for the analysis of proteomic constituents in cancerous cells. The MCF7 breast cancer cell line was chosen as a model system. A series of optimization steps were performed to improve the LC/MS experimental setup, sample preparation, data acquisition and database search protocols, and a data filtering strategy was developed to enable confident identification of a large number of proteins and potential biomarkers. This research has resulted in the identification of >2000 proteins using multiple filtering and p-value sorting. Approximately 1600-1900 proteins had p < 0.001, and, of these, approximately 60% were matched by >or=2 unique peptides. Alternatively, >99% of the proteins identified by >or=2 unique peptides had p < 0.001. When searching the data against a reversed database of proteins, the rate of false positive identifications was 0.1% at the peptide level and 0.4% at the protein level. The typical reproducibility in detecting overlapping proteins across replicate runs exceeded 90% for proteins matched by >or=2 unique peptides. According to their biological function, approximately 200 proteins were involved in cancer-relevant cellular processes, and over 25 proteins were previously described in the literature as putative cancer biomarkers, as they were found to be differentially expressed between normal and cancerous cell states. Among these, biomarkers such PCNA, cathepsin D, E-cadherin, 14-3-3-sigma, antigen Ki-67, TP53RK, and calreticulin were identified. These data were generated by subjecting to MS analysis approximately 42 microg of sample, analyzing 16 SCX peptide fractions, and interpreting approximately 55,000 MS2 spectra. Total MS time required for analysis was 40 h.