Targeted protein quantitation and profiling using PVDF affinity probe and MALDI-TOF MS

Development of a rapid, effective, and highly specific platform for target identification in complex biofluids is one of the most important tasks in proteomic research. Taking advantage of the natural hydrophobic interaction of PVDF with probe protein, a simple and effective method was developed for protein quantitation and profiling. Using antibody-antigen interactions as a proof-of-concept system, the targeted plasma proteins, serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP), could be selectively isolated and enriched from human plasma by antibody-immobilized PVDF membrane and directly identified by MALDI-TOF MS without additional elution step. The approach was successfully applied to human plasma for rapid quantitation and variant screening of SAP, SAA, and CRP in healthy individuals and patients with gastric cancer. The triplexed on-probe quantitative analysis revealed significant overexpression of CRP and SAA in gastric cancer group, consistent with parallel ELISA measurements and pathological progression and prognostic significance reported in previous literatures. Furthermore, the variant mass profiling of the post-translationally modified forms revealed a high occurrence of de-sialic acid SAP in patients with gastric cancer. Due to the versatile assay design, ease of probe preparation without chemical synthesis, and compatibility with MALDI-TOF MS analysis, the methodology may be useful for target protein characterization, functional proteomics, and screening in clinical proteomics.     

Proteome profile of cytosolic component of zebrafish liver generated by LC-ESI MS/MS combined with trypsin digestion and microwave-assisted acid hydrolysis

The zebrafish genome has recently been sequenced and annotated allowing for high-throughput proteomic analysis. Here, we report for the first time a proteomic subset of zebrafish liver, an important organ for metabolizing toxins. Using a newly developed analytical procedure, we have identified 1204 proteins from the cytosolic component of a zebrafish liver tissue sample. Our methods involve cell-compartment fractionation of liver tissue samples, four levels of protein digestion, and off-line two-dimensional liquid chromatography (2-D LC) separations of resultant peptides. Proteins are identified using an electrospray ionization quadrupole time-of-flight tandem mass spectrometer (ESI-QTOF MS/MS), which provides high-resolution and high-accuracy mass measurement of peptide ions and their fragment ions. We demonstrate that greater proteome coverage can be achieved by combining the results obtained from four methods of protein digestion: three tryptic digests (one in buffer, one in methanol, and another in SDS), and a microwave-assisted acid hydrolysate of the protein extracts. Identified proteins--which included several groups of established protein biomarkers--were functionally classified. We discuss the functions and implications of these biomarkers within the context of zebrafish toxicology.     

Quantitative proteome analysis using differential stable isotopic labeling and microbore LC-MALDI MS and MS/MS

We demonstrate an approach for global quantitative analysis of protein mixtures using differential stable isotopic labeling of the enzyme-digested peptides combined with microbore liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Microbore LC provides higher sample loading, compared to capillary LC, which facilitates the quantification of low abundance proteins in protein mixtures. In this work, microbore LC is combined with MALDI MS via a heated droplet interface. The compatibilities of two global peptide labeling methods (i.e., esterification to carboxylic groups and dimethylation to amine groups of peptides) with this LC-MALDI technique are evaluated. Using a quadrupole-time-of-flight mass spectrometer, MALDI spectra of the peptides in individual sample spots are obtained to determine the abundance ratio among pairs of differential isotopically labeled peptides. MS/MS spectra are subsequently obtained from the peptide pairs showing significant abundance differences to determine the sequences of selected peptides for protein identification. The peptide sequences determined from MS/MS database search are confirmed by using the overlaid fragment ion spectra generated from a pair of differentially labeled peptides. The effectiveness of this microbore LC-MALDI approach is demonstrated in the quantification and identification of peptides from a mixture of standard proteins as well as E. coli whole cell extract of known relative concentrations. It is shown that this approach provides a facile and economical means of comparing relative protein abundances from two proteome samples.     

Three-layer sandwich gel electrophoresis: a method of salt removal and protein concentration in proteome analysis

Sample preparation plays a critical role in successful proteomic applications. Features of electrospray mass spectrometry impose limits on the types of buffers, detergents and other reagents that can be used in sample preparation. Unfortunately, many of these mass spectrometry incompatible reagents significantly enhance protein recoveries from complex matrices. This problem prompted our search for a better cleanup protocol. Our data suggest that the Three-layer Sandwich Gel Electrophoresis (TSGE) protocol can solve this problem and provide near quantitative recovery of extremely low concentration proteins from harsh solutions, a feature not available from other cleanup protocols. The hallmark of the TSGE protocol is the combination of the properties of agarose gels (that serve as the matrix to immobilize the proteins of interest) with low- and high-percentage polyacrylamide gels (that serve as the concentration and sealing layers, respectively). By electrophoretically driving the proteins of interest from the agarose matrix into the concentration layer, the TSGE protocol simultaneously concentrates the sample in the concentration layer and provides an environment amenable to downstream buffer exchange and proteolytic digestion. In combination with 2D-LC-MS/MS, the TSGE protocol was evaluated in the analysis of a whole cell extract from the protozoan parasite Toxoplasma gondii. Comparison of our experimental proteomic results with in silico predictions from gene data indicated that TSGE did not bias the protein identification.     

Compositional protein analysis of high density lipoproteins in hypercholesterolemia by shotgun LC-MS/MS and probabilistic peptide scoring

A protein of a biological sample is usually quantified by immunological techniques based on antibodies. Mass spectrometry offers alternative approaches that are not dependent on antibody affinity and avidity, protein isoforms, quaternary structures, or steric hindrance of antibody-antigen recognition in case of multiprotein complexes. One approach is the use of stable isotope-labeled internal standards; another is the direct exploitation of mass spectrometric signals recorded by LC-MS/MS analysis of protein digests. Here we assessed the peptide match score summation index based on probabilistic peptide scores calculated by the PHENYX protein identification engine for absolute protein quantification in accordance with the protein abundance index as proposed by Mann and co-workers (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231-1245). Using synthetic protein mixtures, we demonstrated that this approach works well, although proteins can have different response factors. Applied to high density lipoproteins (HDLs), this new approach compared favorably to alternative protein quantitation methods like UV detection of protein peaks separated by capillary electrophoresis or quantitation of protein spots on SDS-PAGE. We compared the protein composition of a well defined HDL density class isolated from plasma of seven hypercholesterolemia subjects having low or high HDL cholesterol with HDL from nine normolipidemia subjects. The quantitative protein patterns distinguished individuals according to the corresponding concentration and distribution of cholesterol from serum lipid measurements of the same samples and revealed that hypercholesterolemia in unrelated individuals is the result of different deficiencies. The presented approach is complementary to HDL lipid analysis; does not rely on complicated sample treatment, e.g. chemical reactions, or antibodies; and can be used for projective clinical studies of larger patient groups.     

Development and evaluation of a micro- and nanoscale proteomic sample preparation method

Challenges associated with the efficient and effective preparation of micro- and nanoscale (micro- and nanogram) clinical specimens for proteomic applications include the unmitigated sample losses that occur during the processing steps. Herein, we describe a simple "single-tube" preparation protocol appropriate for small proteomic samples using the organic cosolvent, trifluoroethanol (TFE) that circumvents the loss of sample by facilitating both protein extraction and protein denaturation without requiring a separate cleanup step. The performance of the TFE-based method was initially evaluated by comparisons to traditional detergent-based methods on relatively large scale sample processing using human breast cancer cells and mouse brain tissue. The results demonstrated that the TFE-based protocol provided comparable results to the traditional detergent-based protocols for larger, conventionally sized proteomic samples (>100 microg protein content), based on both sample recovery and numbers of peptide/protein identifications. The effectiveness of this protocol for micro- and nanoscale sample processing was then evaluated for the extraction of proteins/peptides and shown effective for small mouse brain tissue samples (approximately 30 microg total protein content) and also for samples of approximately 5000 MCF-7 human breast cancer cells (approximately 500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps.     

A sample preparation process for LC-MS/MS analysis of total protein drug concentrations in monkey plasma samples with antibody

The determination of protein concentrations in plasma samples often provides essential information in biomedical research, clinical diagnostics, and pharmaceutical discovery and development. Binding assays such as ELISA determine meaningful free analyte concentrations by using specific antigen or antibody reagents. Concurrently, mass spectrometric technology is becoming a promising complementary method to traditional binding assays. Mass spectrometric assays generally provide measurements of the total protein analyte concentration. However, it was found that antibodies may bind strongly with the protein analyte such that total concentrations cannot be determined. Thus, a sample preparation process was developed which included a novel "denaturing" step to dissociate binding between antibodies and the protein analyte prior to solid phase extraction of plasma samples and LC-MS/MS analysis. In so doing, the total protein analyte concentrations can be obtained. This sample preparation process was further studied by LC-MS analysis with a full mass range scan. It was found that the protein of interest and other plasma peptides were pre-concentrated, while plasma albumin was depleted in the extracts. This capability of the sample preparation process could provide additional advantages in proteomic research for biomarker discovery and validation. The performance of the assay with the novel denaturing step was further evaluated. The linear dynamic range was between 100.9ng/mL and 53920.0ng/mL with a coefficient of determination (r(2)) ranging from 0.9979 and 0.9997. For LLOQ and ULOQ samples, the inter-assay CV was 12.6% and 2.7% and inter-assay mean accuracies were 103.7% and 99.5% of theoretical concentrations, respectively. For QC samples, the inter-assay CV was between 2.1% and 4.9%, and inter-assay mean accuracies were between 104.1% and 110.0% of theoretical concentrations.     

Digestion protocol for small protein amounts for nano-HPLC-MS(MS) analysis

A miniaturized tryptic digestion protocol for protein analysis has been developed, which works well for small amounts of proteins using small volume of reagents. The protocol starts from 10μL sample volume with total protein content in the low pmol or fmol range (alternatively expressed, in the low ng range). After adding various reagents the total volume of the tryptic digest will increase to 15μL only. This is especially advantageous for nano-HPLC-MS or MALDI analysis which requires (and allows) analysis of few μL aliquots only. Efficiency of the protocol was tested using nano-HLPC-MS(MS). The results show that the developed miniaturized digestion protocol performs at least as well, possibly even better, than conventional protocols using large sample amounts; and is far superior to digestion performed in larger volumes followed by solvent evaporation/resolvation. This is reflected both in signal intensities in MS and in the number of proteins identified by MS/MS.     

Cross-sample validation provides enhanced proteome coverage in rat vocal fold mucosa

The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM)-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high M(r) ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high M(r) glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra) while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match). The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories.     

Prefractionation by digitonin extraction increases representation of the cytosolic and intracellular proteome of Leishmania infantum

Proteome coverage is limited by the dynamic range of proteins present in a sample and often is confined to the analysis of abundant proteins. We have developed a protein prefractionation protocol, based on the differential solubilization of membranes using digitonin, that has allowed an increase in the resolution and depth of comparative proteomic studies. This prefractionation protocol can also be used to infer the subcellular localization of hypothetical proteins as tested experimentally using green fluorescent fusion proteins. The abundant tubulins and associated proteins of the cytoskeleton were removed from the sample using digitonin extraction, hence facilitating the visualization of lower abundance proteins. The digitonin prefractionation protocol was applied for a comparative proteomic analysis of the promastigote and amastigote life cycle stages of Leishmania infantum and has allowed the identification of novel proteins expressed in a stage-specific manner.     

A novel approach and protocol for discovering extremely low-abundance proteins in serum

The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.     

Cost-effective and rapid (3 h) method for combined extraction of DNA,RNA and protein from a single tea leaf sample for genomic and proteomic analysis

This is the first report about the simultaneous extraction of nucleic acids and proteins from tea leaf tissue. Using the present protocol, the DNA, RNA and protein were simultaneously isolated from a single tea leaf sample. The method also maintained the quality and quantity of the isolated biomolecules. The method is cost-effective and takes only 3 h to isolate the starting molecules (DNA, RNA and protein) of central dogma of biology. It was also demonstrated that the isolated DNA, RNA and protein could be successfully used for genomics and proteomic analysis in tea plant which was verified by performing marker study, gene cloning, cDNA preparation, gene expression study and 2-DE.     

Putting the Pieces Together: High-performance LC-MS/MS Provides Network-, Pathway-, and Protein-level Perspectives in Populus

High-performance mass spectrometry (MS)-based proteomics enabled the construction of a detailed proteome atlas for Populus, a woody perennial plant model organism. Optimization of experimental procedures and implementation of current state-of-the-art instrumentation afforded the most detailed look into the predicted proteome space of Populus, offering varying proteome perspectives: (1) network-wide, (2) pathway-specific, and (3) protein-level viewpoints. Together, enhanced protein retrieval through a detergent-based lysis approach and maximized peptide sampling via the dual-pressure linear ion trap mass spectrometer (LTQ Velos), have resulted in the identification of 63,056 tryptic peptides. The technological advancements, specifically spectral-acquisition and sequencing speed, afforded the deepest look into the Populus proteome, with peptide abundances spanning 6 orders of magnitude and mapping to ∼25% of the predicted proteome space. In total, tryptic peptides mapped to 11,689 protein assignments across four organ-types: mature (fully expanded, leaf plastichronic index (LPI) 10–12) leaf, young (juvenile, LPI 4–6) leaf, root, and stem. To resolve protein ambiguity, identified proteins were grouped by sequence similarity (≥ 90%), thereby reducing the protein assignments into 7538 protein groups. In addition, this large-scale data set features the first systems-wide survey of protein expression across different Populus organs. As a demonstration of the precision and comprehensiveness of the semiquantitative analysis, we were able to contrast two stages of leaf development, mature versus young leaf. Statistical comparison through ANOVA analysis revealed 1432 protein groups that exhibited statistically significant (p ≤ 0.01) differences in protein abundance. Experimental validation of the metabolic circuitry expected in mature leaf (characterized by photosynthesis and carbon fixation) compared with young leaf (characterized by rapid growth and moderate photosynthetic activities) strongly testifies to the credibility of the approach. Instead of quantitatively comparing a few proteins, a systems view of all the changes associated with a given cellular perturbation could be made.Mass spectrometry (MS)-based proteomics has experienced tremendous growth in recent years, leading to the establishment of numerous protocols, platforms, and workflows for the characterization of protein expression at the genome level (1). Although these advancements have facilitated comprehensive proteomic investigations of simple bacterial isolates and microbial communities, the application of MS-based proteomics for plants and other higher eukaryotes remains underdeveloped. Recently, large-scale proteomic studies have been directed at characterization of Populus, a woody perennial model organism. With the recent release and subsequent curation of the P. trichocarpa genome (2), these large-scale MS-based proteomic investigations offer the potential to introduce new biological insights into woody perennial plant biology (3, 4, 5). For example, we have recently demonstrated the ability to measure ∼17% of the Populus proteome by coupling multidimensional liquid chromatography (MudPIT)¹ with nano-electrospray tandem mass spectrometry (2D-LC-MS/MS) (6). Relative to the two-dimensional gel-based approaches (7), MudPIT provides enhanced separation and when used in conjunction with MS/MS, surpasses the throughput and number of identifiable proteins detected in complex mixtures (8). Although we have demonstrated the general effectiveness of this approach, the identification and quantitation of the proteins expressed in a plant cell or tissue are still notoriously complicated by a number of factors, including the size and complexity of plant genomes, abundance of protein variants, as well as the dynamic range of protein identification. To overcome these challenges, improvements are needed in sample preparation, MS instrumentation, and data interpretation.The architecture of plant cell walls provides resistance to chemical and biological degradation, thus requiring mechanical and detergent-based lysis for optimal proteome analysis. However, this criterion presents a major challenge for plant proteomic research using electrospray mass spectrometry, as detergent-containing solutions can impede enzymatic digestion and cause significant analyte suppression (9). Therefore, most plant proteomic studies using the “MudPIT” strategy apply mechanical disruption in conjunction with a detergent-free preparation method (10). Typically, strong chaotropic agents such as urea and guanidine hydrochloride are used for the extraction, denaturation, and digestion of proteins. In a recent study, Mann et al. (2009) introduced a filter-aided sample preparation (FASP) method that uses and effectively removes sodium dodecyl sulfate (SDS) before enzymatic digestion and electrospray analysis (11). This study demonstrated enhanced retrieval of peptides from biological materials, yielding a more accurate representation of the proteome. We developed a similar experimental approach for extraction of proteins from plant tissue to obtain a more comprehensive, unbiased proteome characterization well beyond that achievable with currently available methods. Similar to the FASP method, we demonstrate the power of SDS for proteomic sample preparation, not only in its ability to more-thoroughly lyse cells, but also its ability to better solubilize both hydrophilic and hydrophobic proteins. This powerful attribute gives proteolytic enzymes maximum opportunity to generate peptides specific to their cleavage potential so that at least a few representative peptides can be obtained for proteins that would have otherwise been discarded or lost because of insolubility, e.g. membrane-bound proteins. Rather than performing a buffer exchange with urea, depletion of SDS is achieved by precipitating proteins out of solution using trichloroacetic acid.Characterization of protein expression in plants is further complicated by the heterogeneous mixture of various cell types, each with a unique proteome signature and individualized response to environmental chemical or physical signals. This inherent complexity of plant proteomes and the large dynamic range in protein abundance overwhelms current analytical platforms (12). Moreover, biochemical regulatory networks in plants are more elaborate and dynamic than in microbial species; consequently, many biological components are left undiscovered, including modified peptides and low-abundance proteins (13, 14, 15). Recent developments in ion-trap MS instrumentation, namely the dual-pressure linear ion trap mass spectrometer (LTQ Velos), have demonstrated improved ability to comprehensively characterize complex proteomics samples (16). Featuring a newly designed ion source and a two-chamber ion trap mass analyzer, the LTQ Velos achieves greater dynamic range, sensitivity, and speed of spectral acquisition when applied to complex proteomic samples. Cumulatively, the technological advancements afford substantial increases in the detection and identification of both proteins and unique peptides when compared with existing state-of-the-art technologies. Therefore, to satisfy the need for depth of proteome characterization in plants, we apply the newly developed LTQ Velos for mass spectrometry measurements of the Populus proteome.For most terrestrial plants, life begins and ends in the same physical location. For woody perennial plants, this sedentary lifestyle may last thousands of years. One consequence of this lifestyle is that each plant typically experiences dramatic changes in its ambient environment throughout its lifetime and, at any given time, equilibrium between endogenous growth processes and exogenous constraints exerted by the environment must be tightly controlled. To survive under varying environmental conditions, temporal plastic responses evoke patterns of protein expression that progressively influence morphological, anatomical, and functional traits of three principal organs—leaf, root, and stem. Collectively and individually, these organs operate to perceive and respond to periodic and chronic environment conditions. Currently, a comprehensive understanding of the spatial variation in protein expression patterns across the organ types is lacking for woody perennial plants, in which most large-scale proteome analyses with Populus were performed on isolated organs, tissues, organelles, or subcellular structures. For this reason, we combined the state-of-the-art LTQ-Velos platform with the SDS/TCA sample preparation methodology to generate a high-coverage proteome atlas of the principal organ types from Populus.     

The effects of shared peptides on protein quantitation in label-free proteomics by LC/MS/MS

Assessment of differential protein abundance from the observed properties of detected peptides is an essential part of protein profiling based on shotgun proteomics. However, the abundance observed for shared peptides may be due to contributions from multiple proteins that are affected differently by a given treatment. Excluding shared peptides eliminates this ambiguity but may significantly decrease the number of proteins for which abundance estimates can be obtained. Peptide sharing within a family of biologically related proteins does not cause ambiguity if family members have a common response to treatment. On the basis of this concept, we have developed an approach for including shared peptides in the analysis of differential protein abundance in protein profiling. Data from a recent proteomics study of lung tissue from mice exposed to lipopolysaccharide, cigarette smoke, and a combination of these agents are used to illustrate our method. Starting from data where about half of the implicated database protein involved shared peptides, 82% of the affected proteins were grouped into families, based on FASTA annotation, with closure on peptide sharing. In many cases, a common abundance relative to control was sufficient to explain ion-current peak areas for peptides, both unique and shared, that identified biologically related proteins in a peptide-sharing closure group. On the basis of these results, we propose that peptide-sharing closure groups provide a way to include abundance data for shared peptides in quantitative protein profiling by high-throughput mass spectrometry.     

Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.     

Protease inhibitors as possible pitfalls in proteomic analyses of complex biological samples

Sample preparation, especially protein and peptide fractionation prior to identification by mass spectrometry (MS), is typically applied to reduce sample complexity. The second key element in this process is proteolytic digestion, which is performed most often with trypsin. Optimization of this step is an important factor in order to achieve both speed and better performance of proteomic analysis, and tryptic digestion prior to the MS analysis has been a topic of many studies. To date, only a few studies have paid attention to the negative interaction between the proteolytic enzyme and sample components, and sample losses caused by these interactions. In this study, we demonstrated impaired activity after "in solution" tryptic digestion of plasma proteins caused by a potent trypsin inhibitor family, inter-alpha inhibitor proteins. Sample boiling followed by gel electrophoretic separation and "in-gel" digestion drastically improved both the number of identified proteins and the sequence coverage in subsequent LC-ESI-MS/MS. The present investigations show that a thorough validation is necessary when "in solution" digestion followed by LC-MS analysis of complex biological samples is performed. The parallel use of two or more different mass spectrometers can also yield additional information and contribute to further method validation.     

Detection and identification of sub-nanogram levels of protein in a nanoLC-trypsin-MS system

Proteomic workflows involving liquid-based protein separations are an alternative to gel-based protein analysis, however the trypsin digestion procedure is usually difficult to implement, particularly when processing low abundance proteins from capillary column effluent. To convert the protein to peptides for the purpose of identification, current protocols require several sample handling steps, and sample losses become an issue. In this study, we present an improved system that conducts reversed-phase protein chromatography and rapid on-line tryptic digestion requiring sub-nanogram quantities of protein. This system employs a novel mirror-gradient concept that allows for dynamic titration of the column effluent to create optimal conditions for real-time tryptic digestion. The purpose behind this development was to improve the limits of detection of the online concept, to support flow-based alternatives to gel-based proteomics and to simplify the characterization of low abundance proteins. Using test mixtures of proteins, we show that peptide mass fingerprinting with high sequence representation can be easily achieved at the 20 fmol level, with detection limits down to 5 fmol (85 pg myoglobin). Limits of identification using standard data-dependent MS/MS experiments are as low as 10 fmol. These results suggest that the nanoLC-trypsin-MS/MS system could represent an alternative to the conventional "1D-gel to MS" proteomic strategy.     

DeepQuanTR: MALDI‐MS‐based label‐free quantification of proteins in complex biological samples

The quantification of changes in protein abundance in complex biological specimens is essential for proteomic studies in basic and applied research. Here we report on the development and validation of the DeepQuanTR software for identification and quantification of differentially expressed proteins using LC‐MALDI‐MS. Following enzymatic digestion, HPLC peptide separation and normalization of MALDI‐MS signal intensities to the ones of internal standards, the software extracts peptide features, adjusts differences in HPLC retention times and performs a relative quantification of features. The annotation of multiple peptides to the corresponding parent protein allows the definition of a Protein Quant Value, which is related to protein abundance and which allows inter‐sample comparisons. The performance of DeepQuanTR was evaluated by analyzing 24 samples deriving from human serum spiked with different amounts of four proteins and eight complex samples of vascular proteins, derived from surgically resected human kidneys with cancer following ex vivo perfusion with a reactive ester biotin derivative. The identification and experimental validation of proteins, which were differentially regulated in cancerous lesions as compared with normal kidney, was used to demonstrate the power of DeepQuanTR. This software, which can easily be used with established proteomic methodologies, facilitates the relative quantification of proteins derived from a wide variety of different samples.     

Development and optimization of two-dimensional-electrophoresis protocol ofLeptospirillum ferriphilum

Leptospirillum ferriphilum is important in bioleaching, in which process it is often under heavy stresses of heavy metal ions and high oxidation reduction potential (ORP). Two-dimensional-electrophoresis (2-DE) and comparative proteomic analysis are useful to investigate the responses ofL. ferriphilum to environmental stresses. But, 2-DE analysis forL. ferriphilum is not successful as the samples ofL. ferriphilum contain low protein concentration, complex composition, high salt concentration, and many other interfering components, which make it difficult for 2-DE analysis. In this research, optimizations on the sample preparation and purification methods, sample volume, sample loading methods for isoelectric focusing (IEF), and gel visualization methods were made. More than 629 Coomassie stained spots in single gel were obtained. The image quality and protein concentration in most of the spots met the requirements for both differential spots analysis and mass-spectrum analysis. The 2-DE protocol forL. ferriphilum was successfully developed for the first time.     

Simple and robust two-layer matrix/sample preparation method for MALDI MS/MS analysis of peptides

Recent advances in MALDI MS/MS instrumentation allow a high degree of automation in the efficient detection of peptide fragment ions that can be used for protein identification. However, the performance of the technique is dependent on the MALDI sample preparation. We present a simple and robust two-layer sample preparation method tailored for sensitive and reproducible generation of MALDI MS/MS data. This method produces a strong and uniform crystal layer which allows acquisition of high quality MS/MS spectra over the entire sample surface area. Furthermore, due to its crystal strength, the matrix/sample layer can be washed extensively on target, enabling direct analysis of samples containing impurities, such as salts and surfactants. This method is demonstrated to be very useful in routine analysis of in-gel tryptic digests of silver-stained protein gel spots, without the need of desalting steps or hunting for "hot" spots. As an example, seven threonine-phosphorylated proteins involved in signal transduction in response to growth factor stimulation within the lipid raft fractions of the IMR5 neuroblastoma cells have been identified using differential gel display, in-gel digestion and MALDI MS/MS with the new two-layer sample preparation method. Some of these proteins have the functions of maintaining raft structure or cell signaling.