Higher dimensional (Hi-D) separation strategies dramatically improve the potential for cancer biomarker detection in serum and plasma

The plasma proteome has a wide dynamic range of protein concentrations and is dominated by a few highly abundant proteins. Discovery of novel cancer biomarkers using proteomics is particularly challenging because specific biomarkers are expected to be low abundance proteins with normal blood concentrations of low nanograms per milliliter or less. Conventional, one- and two-dimensional proteomic methods including 2D PAGE, 2D DIGE, LC-MS/MS, and LC/LC-MS/MS do not have the capacity to consistently detect many proteins in this range. In contrast, new higher dimensional (Hi-D) separation strategies, utilizing more than two dimensions of fractionation, can profile the low abundance proteome.     

Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay

Oxytocin (OT) is a neuropeptide with an extremely low endogenous level (low pg/ml) in human plasma. It is very challenging to develop a highly sensitive assay to measure endogenous OT, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC–MS/MS) can provide high-throughput and selective methods for quantification of peptides in biological samples. A novel and highly sensitive two-dimensional LC–MS/MS (2D-LC–MS/MS) assay combining solid-phase extraction (SPE) has been developed and validated for the determination of endogenous OT in both human and rat plasma. The lower limit of quantification (LLOQ) was 1.00 pg/ml for human and 50.0 pg/ml for rat. Human plasma diluted with water (1:6, v/v) was successfully optimized as a surrogate matrix for human to prepare standard curves without endogenous interference. The extraction efficiency and absolute recovery were above 65.8% using the HLB SPE procedure, and matrix effects were lower than 12%. The method was validated in the range of 1.00–250 pg/ml for human plasma and 50.0–10,000 pg/ml for rat plasma with precision less than 12.7% and accuracy less than 7%.     

Different immunoaffinity fractionation strategies to characterize the human plasma proteome

Plasma proteins may often serve as indicators of disease and are a rich source for biomarker discovery. However, the intrinsic large dynamic range of plasma proteins makes the analysis very challenging because a large number of low abundance proteins are often masked by a few high abundance proteins. The use of prefractionation methods, such as depletion of higher abundance proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may ultimately prove to be informative biomarkers. But there are few studies on comprehensive investigation of the proteins both in the fractions depleted and remainder. In the present study, two different immunoaffinity fractionation columns for the top-6 or the top-12 proteins in plasma were investigated and both the proteins in column-bound and flow-through fractions were subsequently analyzed. A two-dimensional peptide separation strategy, utilizing chromatographic separation techniques, combined with tandem mass spectrometry (MS/MS) was employed for proteomic analysis of the four fractions. Using the established HUPO PPP criteria, a total of 2401 unique plasma proteins were identified. The Multiple Affinity Removal System yielded 921 and 725 unique proteins from the flow-through and bound fractions, respectively, whereas the Seppro MIXED 12 column yielded identification of 897 and 730 unique proteins from the flow-through and bound fractions, respectively. When more stringent criteria, based on searching against the reversed database, were implemented, 529 unique proteins were identified from the four fractions with the confidence in peptide identification increased from 73.6% to 99%. To determine whether the presence of nontarget proteins in the immunoaffinity-bound fraction could be attributed to their interaction with high abundance proteins, co-immunoprecipitation analysis with an antibody to human plasma albumin was performed, which resulted in an identification of 40 unique proteins from the coimmunoprecipitate with the more stringent criteria. This study illustrated that combining the column-bound and flow-through fractions from immunoaffinity separation affords more extensive profiling of the protein content of human plasma. The presence of nontarget proteins in the column-bound fractions may be induced by their binding to the higher abundance proteins targeted by the immunoaffinity column.     

Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis

Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95-99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein-protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics.     

Comparison of protein enrichment strategies for proteome analysis of plasma

Efforts to discover protein biomarkers in plasma are hampered by the high abundance of few proteins, which interfere with the detection of low‐abundant proteins. Different commercially available protein‐partitioning products were tested for their ability to lower the detection limit of proteins in 2‐D gels. Immuno‐depletion using polyclonal antibodies raised against the proteins of highest abundance (Seppro IgY14 System) was compared with a two‐step immuno‐depletion strategy, where depletion with the Seppro IgY14 column was followed by depletion with the Seppro IgY‐SuperMix system. The third strategy tested was protein pre‐fractionation using the ProteoMiner kit, where proteins compete for binding sites on bead‐bound peptide hexamers with different binding properties. The pre‐fractionated protein samples were analyzed using 2‐DE, which revealed stunning differences in protein patterns. However, detectable protein spots in the different plasma fractions contained exclusively high‐abundant proteins normally present in plasma at concentrations between 1 μg and 40 mg/mL.     

Evaluation of multiprotein immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry

Strategies for removal of high abundance proteins have been increasingly utilized in proteomic studies of serum/plasma and other body fluids to enhance the detection of low abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high abundance protein depletion process still represent common concerns. Here we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system that is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed using this approach, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels but also in a reproducible manner. The results suggest that multiprotein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low abundance proteins in biomarker discovery studies.     

Advances and challenges in liquid chromatography-mass spectrometry-based proteomics profiling for clinical applications

Recent advances in proteomics technologies provide tremendous opportunities for biomarker-related clinical applications; however, the distinctive characteristics of human biofluids such as the high dynamic range in protein abundances and extreme complexity of the proteomes present tremendous challenges. In this review we summarize recent advances in LC-MS-based proteomics profiling and its applications in clinical proteomics as well as discuss the major challenges associated with implementing these technologies for more effective candidate biomarker discovery. Developments in immunoaffinity depletion and various fractionation approaches in combination with substantial improvements in LC-MS platforms have enabled the plasma proteome to be profiled with considerably greater dynamic range of coverage, allowing many proteins at low ng/ml levels to be confidently identified. Despite these significant advances and efforts, major challenges associated with the dynamic range of measurements and extent of proteome coverage, confidence of peptide/protein identifications, quantitation accuracy, analysis throughput, and the robustness of present instrumentation must be addressed before a proteomics profiling platform suitable for efficient clinical applications can be routinely implemented.     

Semi-targeted plasma proteomics discovery workflow utilizing two-stage protein depletion and off-line LC-MALDI MS/MS

A quantitative proteomics workflow was implemented that provides extended plasma protein coverage by extensive protein depletion in combination with the sensitivity and breadth of analysis of two-dimensional LC-MS/MS shotgun analysis. Abundant proteins were depleted by a two-stage process using IgY and Supermix depletion columns in series. Samples are then extensively fractionated by two-dimensional chromatography with fractions directly deposited onto MALDI plates. Decoupling sample fractionation from mass spectrometry facilitates a targeted MS/MS precursor selection strategy that maximizes measurement of a consistent set of peptides across experiments. Multiplexed stable isotope labeling provides quantification relative to a common reference sample and ensures an identical set of peptides measured in the set of samples (set of eight) combined in a single experiment. The more extensive protein depletion provided by the addition of the Supermix column did not compromise overall reproducibility of the measurements or the ability to reliably detect changes in protein levels between samples. The implementation of this workflow is presented for a case study aimed at generating molecular signatures for prediction of first heart attack.     

IgY14 and SuperMix immunoaffinity separations coupled with liquid chromatography-mass spectrometry for human plasma proteomics biomarker discovery

Interest in the application of advanced proteomics technologies to human blood plasma- or serum-based clinical samples for the purpose of discovering disease biomarkers continues to grow; however, the enormous dynamic range of protein concentrations in these types of samples (often >10 orders of magnitude) represents a significant analytical challenge, particularly for detecting low-abundance candidate biomarkers. In response, immunoaffinity separation methods for depleting multiple high- and moderate-abundance proteins have become key tools for enriching low-abundance proteins and enhancing detection of these proteins in plasma proteomics. Herein, we describe IgY14 and tandem IgY14-Supermix separation methods for removing 14 high-abundance and up to 60 moderate-abundance proteins, respectively, from human blood plasma and highlight their utility when combined with liquid chromatography-tandem mass spectrometry for interrogating the human plasma proteome.     

Proteomic analysis of eccrine sweat: implications for the discovery of schizophrenia biomarker proteins

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC-MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC-MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately 2-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC-MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers.     

Simultaneous determination of norethindrone and ethinyl estradiol in human plasma by high performance liquid chromatography with tandem mass spectrometry--experiences on developing a highly selective method using derivatization reagent for enhancing sensitivity

In the present work, for the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous analysis of norethindrone, and ethinyl estradiol, was developed and validated over the concentration range of 50-10000pg/ml and 2.5-500pg/ml, respectively, using 0.5 ml of plasma sample. Norethindrone, ethinyl estradiol, and their internal standards norethindrone-(13)C2, and ethinyl estradiol-d4, were extracted from human plasma matrix with n-butyl chloride. After evaporation of the organic solvent, the extract was derivatized with dansyl chloride and the mixture was injected onto the LC-MS/MS system. The gradient chromatographic elution was achieved on a Genesis RP-18 (50 mm x 4.6 mm, 3 microm) column with mobile phase consisted of acetonitrile, water and formic acid. The flow rate was 1.0 ml/min and the total run time was 5.0 min. Important parameters such as sensitivity, linearity, matrix effect, reproducibility, stability, carry-over and recovery were investigated during the validation. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <6.8% relative standard deviation (RSD) and 4.4% relative error (RE) for norethindrone, and 4.2% RSD and 5.9% RE for ethinyl estradiol, respectively. Chromatographic conditions were optimized to separate analytes of interest from the potential interference peaks, arising from the derivatization. This method could be used for pharmacokinetic and drug-drug interaction studies in human subjects.     

Proteomic profiling of human pleural effusion using two-dimensional nano liquid chromatography tandem mass spectrometry

Pleural effusion, an accumulation of pleural fluid, contains proteins originated from plasma filtrate and, especially when tissues are damaged, parenchyma interstitial spaces of lungs and/or other organs. This study details protein profiles in human pleural effusion from 43 lung adenocarcinoma patients by a two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system. The experimental results revealed the identification of 1415 unique proteins from human pleural effusion. Among these 124 proteins identified with higher confidence levels, some proteins have not been reported in plasma and may represent proteins specifically present in pleural effusion. These proteins are valuable for mass identification of differentially expressed proteins involved in proteomics database and screening biomarker to further study in human lung adenocarcinoma. The significance of the use of proteomics analysis of human pleural fluid for the search of new lung cancer marker proteins, and for their simultaneous display and analysis in patients suffering from lung disorders has been examined.     

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.     

iTRAQ结合2D LC-MS/MS技术在草菇不同生长发育时期蛋白质组分析中的应用

【目的】旨在采用iTRAQ标记结合二维液相色谱串联质谱技术对草菇不同生长发育阶段的差异蛋白质组进行研究。【方法】首先将提取的草菇不同生长阶段蛋白样品进行SDS-PAGE分析,其次将经二维液相色谱串联质谱技术获取的串联质谱数据通过MASCOT软件搜库,之后对鉴定蛋白质数据进行了主成分分析(Principal componentanalysis,PCA)、层次聚类(Hierarchy clustering)分析、K-均值(K-means)聚类和GeneOntology(GO)注释分析。【结果】试验结果显示,共计获得2 335个不同肽段,鉴定到1 039个蛋白质,其中1 030个蛋白质具有定量信息。在子实体阶段中显著上调蛋白质64个,下调蛋白质150个。生物信息学分析表明,iTRAQ标记技术结合二维液相色谱串联质谱可对不同生长发育时期的草菇蛋白样品进行有效地分离和鉴定。【结论】这一研究结果为深入研究草菇乃至其他大型担子菌子实体形成和发育的分子机制提供借鉴。     

草菇子实体不同成熟阶段的比较蛋白质组学分析

采用iTRAQ标记结合二维液相色谱串联质谱技术对草菇不同成熟阶段的差异蛋白质组进行研究。首先将提取的草菇不同成熟阶段蛋白样品进行SDS-PAGE分析,其次将经二维液相色谱串联质谱技术获取的串联质谱数据通过MASCOT软件搜库,之后对鉴定蛋白质数据进行了KEGG代谢通路分析。试验共计获得2 335个不同肽段, 鉴定到1 039个蛋白质,其中1 030个蛋白质具有定量信息。与蛋形期相比,在伸长期和成熟期阶段显著上调蛋白质85个,下调蛋白质103个。KEGG代谢通路分析结果显示,草菇不同成熟阶段中的68个差异蛋白质可定位于4种伞菌目模式真菌（灰盖鬼伞、双色蜡蘑、可可丛枝病菌和裂褶菌）的45个不同生物代谢途径,全景展示出草菇成熟阶段差异表达蛋白质定位的代谢网络。结果表明,iTRAQ标记技术结合二维液相色谱串联质谱可对不同生长发育时期的草菇蛋白样品进行有效地分离和鉴定。     

Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy

Currently available radioimmunoassay methods for estradiol in serum lack sufficient sensitivity and precision to monitor estradiol levels in patients placed on third generation aromatase inhibitors. We recently validated a gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol and determined estrogen levels in normal post-menopausal women and in women with breast cancer before and during administration of aromatase inhibitors. Validation of the GC/MS/MS assay in human plasma and human serum included determination of assay sensitivity (<0.63 pg/ml), precision (all CVs less than 17.8%), recovery (98-103%), and linearity of recovery (R=0.998). Levels of estradiol were lower when assayed by GC/MS/MS compared to RIA under all conditions (7.26+/-4.82 pg/ml versus 11.9+12.0 pg/ml in normal post-menopausal women; 5.88+/-3.43 pg/ml versus 13.8+/-7.5 pg/ml in breast cancer patients prior to treatment; and<0.63 pg/ml versus 5.8+/-4.1 pg/ml during aromatase inhibitor therapy). Fifty-five women treated either with atamestane/toremiphene or letrozole/placebo were monitored for estradiol levels at 4, 8 and 12 weeks of therapy. The mean levels of estradiol during aromatase inhibitor therapy was 5.8+/-4.1 pg/ml as measured by RIA and <0.63 pg/ml by GC/MS/MS. The degree of suppression with the aromatase inhibitors as detected by RIA was 58% versus >89% by GC/MS. These results suggest that most RIA methods detect cross-reacting estrogen metabolites and yield higher measured levels than GC/MS/MS. Several pharmacological and clinical considerations suggest that GC/MS/MS should become the preferred method for monitoring aromatase inhibitor therapy.     

Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry

Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (< 100 microg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 microg) and small (10 microg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 microg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 microg) samples. In nine replicate analyses of tryptic peptides from 50 microg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens.     

Developing a strong anion exchange/RP (SAX/RP) 2D LC system for high‐abundance proteins depletion in human plasma

Human plasma is dominated by high‐abundance proteins which severely impede the detection of low‐abundance proteins. Unfortunately, now there is no efficient method for large‐scale depletion of high‐abundance proteins in human plasma. In this study, we developed a new strategy, strong anion exchange (SAX)/RP 2D LC system, which has potential for large‐scale depletion of high‐abundance proteins in human plasma. Separation gradients of the system were optimized to ensure an extensive separation of plasma proteins. Plasma was fractionated into 67 fractions by SAX. All these fractions were subjected a thorough separation by the 2D RPLC and 66 peaks with high UV absorption (>20 mAU) at 215 nm were collected. Proteins in these peaks were identified by LC‐MS/MS analysis. Results showed that 83 proteins could be identified in these peaks, 68 among them were reported to be high‐ or middle‐abundance proteins in plasma. All these proteins had definite retention times and were mapped in the 2D SAX‐RP system, which resulted in accurate depletion of high‐abundance proteins with ease. Our studies provide a convenient and effective method for large‐scale depletion of high‐abundance proteins and in‐depth research in human plasma proteomics.     

The chicken egg yolk plasma and granule proteomes

Using 1-D SDS-PAGE, LC-MS/MS, and MS(3), we identified 119 proteins from chicken egg yolk, 86 of which were not identified in yolk previously. Proteins were roughly quantitated by calculating their exponentially modified protein abundance index (emPAI) to classify them as major or minor yolk components, and to estimate their distribution between yolk plasma and yolk granular fraction. The proteins with highest abundance were serum albumin, the vitellogenin cleavage products, apovitellenins, IgY, ovalbumin, and 12 kDa serum protein with cross-reactivity to beta2-microglobulin. In addition yolk contained many other serum and egg white proteins, the proteases nothepsin and thrombin, numerous protease inhibitors, and antioxidative enzymes, such as superoxide dismutase and glutathione peroxidase. Among the moderately abundant proteins were two alpha2-macroglobulin-like proteins different from egg white alpha2-macroglobulin, and the major biotin-binding protein of yolk. An unexpected identification was that of the eggshell matrix protein ovocleidin-116, which was previously thought to be eggshell-specific. The list of chicken egg yolk proteins provided in this report is by far the most comprehensive at present and may serve as a starting point for the characterization of less well-known yolk proteins.     

Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins

Quantitative LC-MS/MS assays were designed for tryptic peptides representing 53 high and medium abundance proteins in human plasma using a multiplexed multiple reaction monitoring (MRM) approach. Of these, 47 produced acceptable quantitative data, demonstrating within-run coefficients of variation (CVs) (n = 10) of 2-22% (78% of assays had CV <10%). A number of peptides gave CVs in the range 2-7% in five experiments (10 replicate runs each) continuously measuring 137 MRMs, demonstrating the precision achievable in complex digests. Depletion of six high abundance proteins by immunosubtraction significantly improved CVs compared with whole plasma, but analytes could be detected in both sample types. Replicate digest and depletion/digest runs yielded correlation coefficients (R(2)) of 0.995 and 0.989, respectively. Absolute analyte specificity for each peptide was demonstrated using MRM-triggered MS/MS scans. Reliable detection of L-selectin (measured at 0.67 microg/ml) indicates that proteins down to the microg/ml level can be quantitated in plasma with minimal sample preparation, yielding a dynamic range of 4.5 orders of magnitude in a single experiment. Peptide MRM measurements in plasma digests thus provide a rapid and specific assay platform for biomarker validation, one that can be extended to lower abundance proteins by enrichment of specific target peptides (stable isotope standards and capture by anti-peptide antibodies (SISCAPA)).