Serum protein profiling by miniaturized solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

Serum profiling by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) holds promise as a clinical tool for early diagnosis of cancer and other human diseases. Sample preparation is key to achieving reproducible and well-resolved signals in MALDI-MS; a prerequisite for translation of MALDI-MS based diagnostic methods to clinical applications. We have investigated a number of MALDI matrices and several miniaturized solid-phase extraction (SPE) methods for serum protein concentration and desalting with the aim of generating reproducible, high-quality protein profiles by MALDI-MS. We developed a simple protocol for serum profiling that combines a matrix mixture of 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid with miniaturized SPE and MALDI-MS. Functionalized membrane discs with hydrophobic, ion-exchange or chelating properties allowed reproducible MALDI mass spectra (m/z 1000-12,000) to be obtained from serum. In a proof-of-principle application, SPE with chelating material and MALDI-MS identified protein peaks in serum that had been previously reported for distinguishing a person diagnosed with breast cancer from a control. These preliminary results indicate that this simple SPE/MALDI-MS method for serum profiling provides a versatile and scalable platform for clinical proteomics.     

基于共价色谱分离的含半胱氨酸肽段富集策略

多维色谱分离、串联质谱分析技术已在蛋白质组研究中得到广泛应用。然而生物样品的蛋白质以及全酶切肽段具有高度的复杂性,这严重干扰了蛋白质高通量、规模化的分析。通过标签肽段富集进行样品预分离可以降低体系的复杂程度。本文建立了一种基于共价色谱技术选择性分离富集含半胱氨酸肽的方法,从而降低了样品体系的复杂程度。首先以牛血清白蛋白(BSA)的酶切肽段为模型,对富集条件进行了优化和考察,并在此基础上通过5种蛋白质酶切肽段混合物的富集对该方法进行了验证。结果证明此方法的重现性好,富集效率高,富集特异性好,能有效地富集鉴定含半胱氨酸肽段。所建立的方法在复杂体系的蛋白质组研究中具有广泛的应用前景,为复杂样品的蛋白质高通量、自动化、规模化鉴定和定量研究提供了实用技术。     

Reproducibility of serum protein profiling by systematic assessment using solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

Protein profiling of human serum by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is potentially a new diagnostic tool for early detection of human diseases, including cancer. Sample preparation is a key issue in MALDI MS and the analysis of complex samples such as serum requires optimized, reproducible methods for handling and deposition of protein samples. Data acquisition in MALDI MS is also a critical issue, since heterogeneity of sample deposits leads to attenuation of ion signals in MALDI MS. In order to improve the robustness and reproducibility of MALDI MS for serum protein profiling we investigated a range of sample preparation techniques and developed a statistical method based on repeated analyses for evaluation of protein-profiling performance of MALDI MS. Two different solid-phase extraction (SPE) methods were investigated, namely custom-made microcolumns and commercially available magnetic beads. Using these two methods, nineteen different sample preparation methods for serum profiling by MALDI MS were systematically tested with regard to matrix selection, stationary phase, selectivity, and reproducibility. Microcolumns were tested with regard to chromatographic properties; reversed phase (C8, C18, SDB-XC), ion-exchange (anion, weak cation, mixed-phase (SDB-RPS)) and magnetic beads were tested with regard to chromatographic properties; reversed phase (C8) or affinity chromatography (Cu-IMAC). The reproducibility of each sample preparation method was determined by enumeration and analysis of protein signals that were detected in at least six out of nine spectra obtained by three triplicate analyses of one serum sample.A candidate for best overall performance as evaluated by the number of peaks generated and the reproducibility of mass spectra was found among the tested methods. Up to 418 reproducible peaks were detected in one cancer serum sample. These protein peaks can be part of a possible diagnostic profile, suggesting that this sample preparation method and data acquisition approach is suitable for large-scale analysis of serum samples for protein profiling.     

Multidimensional separation of tryptic peptides from human serum proteins using reversed‐phase,strong cation exchange,weak anion exchange,and fused‐core fluorinated stationary phases

Proteome profiling of crude serum is a challenging task due to the wide dynamic range of protein concentrations and the presence of high‐abundance proteins, which cover >90% of the total protein mass in serum. Peptide fractionation on strong cation exchange, weak anion exchange in the electrostatic repulsion hydrophilic interaction chromatography (ERLIC) mode, RP C₁₈ at pH 2.5 (low pH), fused‐core fluorinated at pH 2.5, and RP C₁₈ at pH 9.7 (high pH) stationary phases resulted in two to three times more identified proteins and three to four times more identified peptides in comparison with 1D nanoChip‐LC–MS/MS quadrupole TOF analysis (45 proteins, 185 peptides). The largest number of peptides and proteins was identified after prefractionation in the ERLIC mode due to the more uniform distribution of peptides among the collected fractions and on the RP column at high pH due to the high efficiency of RP separations and the complementary selectivity of both techniques to low‐pH RP chromatography. A 3D separation scheme combining ERLIC, high‐pH RP, and low‐pH nanoChip‐LC–MS/MS for crude serum proteome profiling resulted in the identification of 208 proteins and 1088 peptides with the lowest reported concentration of 11 ng/mL for heat shock protein 74.     

Toward high sequence coverage of proteins in human breast cancer cells using on-line monolith-based HPLC-ESI-TOF MS compared to CE MS

A method is developed toward high sequence coverage of proteins isolated from human breast cancer MCF10 cell lines using a 2-D liquid separations. Monolithic-capillary columns prepared by copolymerizing styrene with divinylbenzene are used to achieve high-resolution separation of peptides from protein digests. This separation is performed with minimal sample preparation directly from the 2-D liquid fractionation of the cell lysate. The monolithic column separation is directly interfaced to ESI-TOF MS to obtain a peptide map. The protein digests were also analyzed by MALDI-TOF MS and an accurate M(r) of the intact protein was obtained using an HPLC-ESI-TOF MS. The result is that these techniques provide complementary information where nearly complete sequence coverage of the protein is obtained and can be compared to the experimental M(r) value. The high sequence coverage provides information on isoforms and other post-translational modifications that would not be available from methods that result in low sequence coverage. The results from the use of monolithic columns are compared to that obtained by CE-MS. The monolithic column separations provide a rugged and highly reproducible method for separating protein digests prior to MS analysis and is suited to confidently identify biomarkers associated with cancer progression.     

Nanodiamond-based two-step sampling of multiply and singly phosphorylated peptides for MALDI-TOF mass spectrometry analysis

Simultaneous detection of multiply and singly phosphorylated peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging because of suppression effects during ionization. In oder to overcome this problem, this study presents a new approach to improve the detection of phosphopeptides by stepwise enrichment using polyarginine-coated (PA-coated) and titanium dioxide-coated (TiO(2)-coated) nanodiamonds for fractionation of multiply and singly phosphorylated peptides prior to on-probe MALDI MS analysis. The feasibility of this approach was demonstrated using synthetic peptides containing different numbers of phosphate groups, tryptic digests of α-casein, β-casein, and complex protein mixtures. The high specificity of the approach is shown in its effective enrichment and fractionation of phosphopeptides from the digest of β-casein and bovine serum albumin at a molar ratio as low as 1 : 1000, which out-performs the commercial Fe(3+)-IMAC and TiO(2) isolation kits. It offers a simple and effective alternative for the fractionation and identification of multiply and singly phosphorylated peptides by MALDI MS and allows for deduction of more information from limited starting materials.     

Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate-protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate-spacer-carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate.     

Serum protein profiling in patients with inflammatory bowel diseases using selective solid-phase bulk extraction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemometric data analysis

The identification of new biomarkers or a disease-related protein fingerprint for inflammatory bowel diseases (IBDs) represents a major task in the diagnosis, prognosis and pharmacological therapy. To address these issues, a simple and rapid analytical proteomic method for serum protein profiling based on selective beads-based solid-phase bulk extraction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and chemometric data analysis was developed. Serum proteins from healthy subjects (22) and patients with Crohn's disease (15) and ulcerative colitis (26) were selectively extracted according to reversed-phase (C18), strong anion-exchange (SAX) and metal ion affinity (IDA-Cu(II)) principles. This approach allowed enrichment of serum proteins/peptides due to the high interaction surface between analytes and the solid phase and high recovery due to the elution step performed directly on the MALDI-target plate. The MALDI-TOF MS serum protein profiles were acquired and, after a data pre-processing step, analyzed by linear discriminant analysis (LDA), a chemometric classification technique, in order to classify serum samples among healthy subjects and patients with inflammatory bowel diseases (IBDs). Since the high number of variables in the MALDI spectra (more than 16000 m/z values) prevents the use of LDA, the variables were reduced to 10-20 by features selection, thus allowing the evaluation of a pattern of m/z values with high discriminant power. Serum protein profiles obtained by reversed-phase extraction and the selection of 20 m/z values gave the best overall prediction ability (96.9%). The recognition of these m/z values may allow the identification of protein biomarkers involved in IBDs.     

Discrimination of Penicillium isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral 'fingerprints' for twelve Penicillium species. Prior to MALDI-TOF MS analysis, eight replicate cultures of each Penicillium species were subjected to three one-minute bead-beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contained abundant peaks in the range of m/z 5000-20 000, and allowed unambiguous discrimination between species. In addition, a biomarker common to all Penicillium mass spectra was observed at m/z 13 900. Discriminant analysis using the MALDI-TOF MS data yielded classification error rates of 0% (i.e. 100% correct identification), indicating that MALDI-TOF MS data may be a useful diagnostic tool for the objective identification of Penicillium species of environmental and clinical importance.     

Material-enhanced laser desorption/ionization (MELDI)—A new protein profiling tool utilizing specific carrier materials for time of flight mass spectrometric analysis

Over the past couple of years, proteomics pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian, breast, or prostate cancer, without identification of single biomarkers. MALDI-TOF MS, for example, offers a simple approach for fast and reliable protein profiling, especially by using carrier materials with various physical and chemical properties, in combination with a MALDI matrix. This approach is referred to as material-enhanced laser desorption/ionization (MELDI). In this paper, we report the development and application of derivatized carrier materials [cellulose, silica, poly(glycidyl methacrylate/divinylbenzene) (GMA/DVB) particles, and diamond powder] for fast and direct MALDI-TOF MS protein profiling. The applicability of MELDI for rapid protein profiling was evaluated with human serum samples. These carriers, having various hydrophobicities, resulted in characteristic mass fingerprints, even if all materials were derivatized with iminodiacetic acid (IDA) to yield an immobilized metal affinity chromatography (IMAC) functionality. Our study demonstrates that analyzing complex biological samples, such as human serum, by employing different MELDI carrier materials yielded type- and size-dependent performance variation.     

Proteome analysis of human dorsolateral prefrontal cortex using shotgun mass spectrometry

The prefrontal cortex executes important functions such as differentiation of conflicting thoughts, correct social behavior and personality expression, and is directly implicated in different neurodegenerative diseases. We performed a shotgun proteome analysis that included IEF fractionation, RP-LC, and MALDI-TOF/TOF mass spectrometric analysis of tryptic digests from a pool of seven human dorsolateral prefrontal cortex protein extracts. In this report, we present a catalog of 387 proteins expressed in these samples, identified by two or more peptides and high confidence search scores. These proteins are involved in different biological processes such as cell growth and/or maintenance, metabolism/energy pathways, cell communication/signal transduction, protein metabolism, transport, regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism, and immune response. This analysis contributes to the knowledge of the human brain proteome by adding sample diversity and protein expression data from an alternative technical approach. It will also aid comparative studies of different brain areas and medical conditions, with future applications in basic and clinical research.     

Knowledge-based proteome profiling: considering identified proteins to evaluate separation efficiency by 2-D PAGE

Proteome profiling techniques rely on the separation of proteins or peptides and their subsequent quantification. The reliability of this technique is still limited because a proteome profiling result does not necessarily represent the true protein composition of the analysed sample, thus seriously hampering proper data interpretation. Many experimentally observed proteome alterations are biologically not significant. It was the aim of this study to use the knowledge of the biological context of proteins in order to establish optimised proteome profiling protocols. While 2-D spot patterns of total cell protein fractions were found to poorly represent the true protein composition, purified subcellular protein fractions were found to better represent the protein composition of the analysed sample. The application of a standardised protocol to different kinds of cells revealed several striking observations. Firstly, the protein composition of cultured cells of various origins is very similar. Secondly, proteome alterations observed with the described protocols do make sense from a biologic point of view and may thus be considered as truly representative for the analysed samples. Thirdly, primary white blood cells isolated from different donors were found to show minor, but reproducible and significant individual differences. We designate the consideration of known properties of identified proteins in proteome profiles as a knowledge-based approach. The present data suggest that this approach may tremendously help to improve the applied techniques and assess the results. We demonstrate that the fulfilment of well-defined criteria of proteome profiles eventually results in reliable and biologically relevant data.     

In-tip nanoreactors for cancer cells proteome profiling

Mass spectrometry (MS)-based proteome profiling is essential for molecular diagnostics in modern biomedical study. To date, sample preparation including protein extraction and proteolysis is still very challenging and lack of efficiency. Recently tips-based sample preparation protocols exhibit strong potentials to achieve the goal of “a proteome in an hour”. However, in-tip proteolysis is still rarely reported and far from ideal for dealing with complex bio-samples. In this work, nanoreactors encapsulated micropipette tips were demonstrated as high performance devices for fast (∼minutes) and multiplexing proteolysis to assist the profiling of cancer cells proteome. Nanoporous silica materials with controlled pore size and surface chemistry were prepared as nanoreactors and encapsulated in micropipette tips for efficient in situ proteolysis. The as-constructed device showed desirable sensitivity (LOD of 0.204 ± 0.008 ng/μL and LOQ of 0.937 ± 0.055 ng/μL), selectivity, stability (two months under −20 °C), reusability (at least 10 times), and little memory effect in MS based bottom-up proteomic analysis. It was used for comprehensive protein mapping from cancer cell lines. The number of identified proteins was increased by 18%, 22%, 52%, and 52% dealing with HepG2, F56, MCF7, and HCCLM3 cancer cells, compared to traditional in-solution proteolysis based bottom-up proteomic strategy. With the enhanced performance, our work built a novel, efficient and miniaturized platform for facile proteomic sample preparation, which is promising for advanced biomarkers discovery in biomedical study.     

Zirconia layer coated mesoporous silica microspheres as HILIC SPE materials for selective glycopeptide enrichment

Characterization of protein glycosylation requires highly specific methods for the enrichment of glycopeptides because of their sub-stoichiometric glycosylation-site occupancy. The hydrophilic affinity based strategy has attracted more attention, owing to its broad glycan specificity, good reproducibility, and compatibility with mass spectrometric (MS) analysis. Several polar matrices have emerged for hydrophilic interaction chromatography (HILIC) approaches, including sepharose, cellulose, ZIC-HILIC and titania. Here, we present the solid-phase extraction (SPE) utility of zirconia coated mesoporous silica (ZrO(2)/MPS) microspheres for glycopeptide isolation prior to MS analysis. The high specificity of this SPE approach was demonstrated by the enrichment of glycopeptides from the digests of model glycoproteins in HILIC mode. ZrO(2)/MPS microspheres show superior selectivity and glycosylation heterogeneity coverage for glycopeptide enrichment to conventional sepharose. Furthermore, digested mixtures of the phosphoprotein α-casein and IgG were also treated with ZrO(2)/MPS HILIC SPE materials, which exhibited that glycopeptides could be effectively enriched with interference from phosphorylated peptides.     

Comparison of surfactant-assisted shotgun methods using acid-labile surfactants and sodium dodecyl sulfate for membrane proteome analysis

Three surfactant-assisted shotgun methods using acid labile surfactants, sodium-3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)-methoxyl]-1-propanesulfonate (RapiGest) and 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), and sodium dodecyl sulfate (SDS) were investigated for their applicability to membrane proteome analysis. It is shown that RapiGest is a preferred reagent for handling membrane proteomes of Escherichia coli and MCF7 cells for liquid chromatography tandem mass spectrometry (LC MS/MS) analysis of tryptic digests. The RapiGest method allowed identification of more peptides and proteins than the SDS and PPS methods and there was no apparent bias for the type of peptides and proteins identified by the RapiGest and SDS methods, while a slightly higher proportion of hydrophilic peptides and proteins were identified by the PPS method. The performance of the SDS and PPS methods is similar in terms of the numbers of peptides and proteins identified. Since the SDS method required the removal of SDS using a technique such as strong-cation exchange (SCX), we further investigated the effect of SCX on sample loss through analyzing the digest of an enriched E. coli membrane fraction as well as a standard protein, bovine serum albumin (BSA). The results showed that extensive sample loss (as much as 62%) was encountered during the SCX cleaning step. We then applied the RapiGest method in combination with two-dimensional LC MS/MS to characterize the E. coli membrane proteome. In total, 1626 unique proteins (5799 unique peptides) were identified with a peptide false discovery rate of 2.4%. About 60% of the identified proteins with known cellular locations were found to be membrane proteins. Among them, about 75% were integral membrane proteins. This work represents one of the most comprehensive profiles of E. coli membrane proteome generated by a proteomic technique.     

A metabolomic protocol for plant systematics by matrix-assisted laser-desorption/ionization time-of flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS) has been widely used for the identification and classification of microorganisms based on their proteomic fingerprints. However, the use of MALDI-TOF MS in plant research has been very limited. In the present study, a first protocol is proposed for metabolic fingerprinting by MALDI-TOF MS using three different MALDI matrices with subsequent multivariate data analysis by in-house algorithms implemented in the R environment for the taxonomic classification of plants from different genera, families and orders. By merging the data acquired with different matrices, different ionization modes and using careful algorithms and parameter selection, we demonstrate that a close taxonomic classification can be achieved based on plant metabolic fingerprints, with 92% similarity to the taxonomic classifications found in literature. The present work therefore highlights the great potential of applying MALDI-TOF MS for the taxonomic classification of plants and, furthermore, provides a preliminary foundation for future research.     

A proteome signature for intrauterine growth restriction derived from multifactorial analysis of mass spectrometry-based cord blood serum profiling

Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity and mortality, thus contributing substantially to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the control group (n = 15) to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by MALDI-TOF mass spectrometric profiling. The six best differentiating ion signals at m/z 8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of this proteome signature, apolipoprotein C-III(0) , a derivative lacking glycosylation, has been found more abundant in the IUGR cord blood serum samples, irrespective of gestational age. Hence, we suggest apolipoprotein C-III(0) as potential key-marker of the here proposed IUGR proteome signature, as it is a very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.     

Characterisation and evaluation of metal-loaded iminodiacetic acid-silica of different porosity for the selective enrichment of phosphopeptides

Silica particles of different porosity were functionalised with iminodiacetic acid (IDA) and loaded with Fe(III) to yield immobilised metal affinity chromatography stationary phases (Fe(III)-IDA-silica) for phosphopeptide enrichment. The elution step of bound phosphopeptides was optimised with a 32P radioactive labelled peptide by a comprehensive study. Several elution systems, including phosphate buffers of different pH and concentration and ethylenediaminetetraacetic acid solutions were employed. Furthermore the effect of support porosity on elution behaviour was investigated. Under best conditions recoveries higher than 90% were achieved. A solid-phase extraction (SPE) protocol was developed for fractionation of phosphorylated and non-phosphorylated peptides and desalting of the fractions which is essential for subsequent mass spectrometric analysis by the combination of Fe(III)-IDA-silica and C18-silica particles. The pH of the loading buffer was found to be a critical parameter for the efficiency of the SPE protocol. As tryptic digests of alpha-lactalbumin, lysozyme and ribonuclease A mixed with three synthetic phosphopeptides were fractionated, pH 2.5 provided minimal proportion of unspecific bound peptides when comparing the fractions after mu-LC-electrospray ionization MS separation. The effect of a sample derivatisation reaction (methylation) on the efficiency of phosphopeptide enrichment was further investigated. Blocking carboxylate groups by methyl ester formation totally prevented unspecific interaction with the immobilised Fe(III) ions, but generated partially methylated phosphopeptides that increased the complexity of the phosphorylated fraction.     

A magnetic bead‐based serum proteomic fingerprinting method for parallel analytical analysis and micropreparative purification

ProteinChip surface‐enhanced laser desorption/ionization technology and magnetic beads‐based ClinProt system are commonly used for semi‐quantitative profiling of plasma proteome in biomarker discovery. Unfortunately, the proteins/peptides detected by MS are non‐recoverable. To obtain the protein identity of a MS peak, additional time‐consuming and material‐consuming purification steps have to be done. In this study, we developed a magnetic beads‐based proteomic fingerprinting method that allowed semi‐quantitative proteomic profiling and micropreparative purification of the profiled proteins in parallel. The use of different chromatographic magnetic beads allowed us to obtain different proteomic profiles, which were comparable to those obtained by the ProteinChip surface‐enhanced laser desorption/ionization technology. Our assays were semi‐quantitative. The normalized peak intensity was proportional to concentration measured by immunoassay. Both intra‐assay and inter‐assay coefficients of variation of the normalized peak intensities were in the range of 4–30%. Our method only required 2 μL of serum or plasma for generating enough proteins for semi‐quantitative profiling by MALDI‐TOF‐MS as well as for gel electrophoresis and subsequent protein identification. The protein peaks and corresponding gel spots could be easily matched by comparing their intensities and masses. Because of its high efficiency and reproducibility, our method has great potentials in clinical research, especially in biomarker discovery.     

Non-invasive detection of drug toxicity in rats by solid-phase extraction and MALDI-TOF analysis of urine samples

An access to fast and non-invasive techniques to infer or predict the drug-induced injury caused by newly developed drugs and to monitor therapeutic efficacy of established drugs during treatment are of the outmost importance in pharmaceutical industry and clinical diagnosis. Peptidome and low molecular weight proteome profiling is an emerging technique that allows the recognition of distinctive patterns and differentiation among diverse physiopathological conditions. In this article, we evaluated the utility of peptide/small protein profiling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with WCX magnetic bead-based solid-phase extraction as a screening tool for drug toxicity assessment in urine samples. Given that drug-induced injury is primarily reflected in liver, three different, well-described hepatotoxic drugs were chosen for this work. These were: carbon tetrachloride (CCl₄) which induces liver fibrosis, d(+)-galactosamine as a model for acute liver injury, and Escherichia coli-derived lipopolysaccharide to study the damage caused by endotoxins. The profiles obtained with a correct clustering analysis show that this methodology can be used as a non-invasive and straightforward approach to test for potential drug toxicity. Pharmaceutical research and drug development studies could benefit from this methodology as liver injury inducer compounds could be easily detected in vivo by non-invasive means, accelerating the launch of safer drugs to the market.