High-coverage quantitative proteomics using amine-specific isotopic labeling

Peptide dimethylation with isotopically coded formaldehydes was evaluated as a potential alternative to techniques such as the iTRAQ method for comparative proteomics. The isotopic labeling strategy and custom-designed protein quantitation software were tested using protein standards and then applied to measure proteins levels associated with Alzheimer's disease (AD). The method provided high accuracy (10% error), precision (14% RSD) and coverage (70%) when applied to the analysis of a standard solution of BSA by LC-MS/MS. The technique was then applied to measure protein abundance levels in brain tissue afflicted with AD relative to normal brain tissue. 2-D LC-MS analysis identified 548 unique proteins (p<0.05). Of these, 349 were quantified with two or more peptides that met the statistical criteria used in this study. Several classes of proteins exhibited significant changes in abundance. For example, elevated levels of antioxidant proteins and decreased levels of mitochondrial electron transport proteins were observed. The results demonstrate the utility of the labeling method for high-throughput quantitative analysis.     

Multidimensional liquid phase protein separations in conjunction with stable isotope labelling for quantitative proteomics

A method for quantitative protein profiling has been developed utilising multidimensional liquid phase protein separations in conjunction with stable isotope labelling. This approach combines the advantages of high throughput, automated, reproducible protein separations with accurate protein quantitation performed in the mass spectrometer. Escherichia coli cells were grown in the presence and absence of the DNA methylation inhibitor 5-Azacytidine on 14N and 15N enriched media. Protein separations were performed using ion exchange chromatography in the first dimension and RP capillary chromatography in the second dimension. UV absorbance measurements were used for the initial semiquantitative identification of differentially expressed proteins. Selected peaks from the mixed 15N/14N lysates were used for the accurate quantitation performed in the mass spectrometer using the ratios of the stable isotopes. Using this approach, a number of differentially expressed proteins have been identified. Moreover, this approach overcomes a number of caveats associated with multidimensional liquid phase protein separations, including the presence of multiple proteins present in a single chromatographic peak.     

Reference map for liquid chromatography–mass spectrometry-based quantitative proteomics

The accurate mass and time (AMT) tag strategy has been recognized as a powerful tool for high-throughput analysis in liquid chromatography–mass spectrometry (LC–MS)-based proteomics. Due to the complexity of the human proteome, this strategy requires highly accurate mass measurements for confident identifications. We have developed a method of building a reference map that allows relaxed criteria for mass errors yet delivers high confidence for peptide identifications. The samples used for generating the peptide database were produced by collecting cysteine-containing peptides from T47D cells and then fractionating the peptides using strong cationic exchange chromatography (SCX). LC–tandem mass spectrometry (MS/MS) data from the SCX fractions were combined to create a comprehensive reference map. After the reference map was built, it was possible to skip the SCX step in further proteomic analyses. We found that the reference-driven identification increases the overall throughput and proteomic coverage by identifying peptides with low intensity or complex interference. The use of the reference map also facilitates the quantitation process by allowing extraction of peptide intensities of interest and incorporating models of theoretical isotope distribution.     

Systematic research on the pretreatment of peptides for quantitative proteomics using a C18 microcolumn

Reversed phase microcolumns have been widely used for peptide pretreatment to desalt and remove interferences before tandem LC–MS in proteomics studies. However, few studies have characterized the effects of experimental parameters as well as column characteristics on the composition of identified peptides. In this study, several parameters including the concentration of ACN in washing buffer, the microcolumn's purification effect, the peptide recovery rate, and the dynamic‐binding capacity were characterized in detail, based upon stable isotope labeling by amino acids in a cell culture quantitative approach. The results showed that peptide losses can be reduced with low ACN concentration in washing buffers resulting in a recovery rate of approximately 82%. Furthermore, the effects of ACN concentration and loading amount on the properties of identified peptides were also evaluated. We found that the dynamic‐binding capacity of the column was approximately 26 μg. With increased loading amounts, more hydrophilic peptides were replaced by hydrophobic peptides.     

Evaluation of comprehensive multidimensional separations using reversed-phase, reversed-phase liquid chromatography/mass spectrometry for shotgun proteomics

Two-dimensional liquid-chromatographic (LC) separation followed by mass spectrometric (MS) analysis was examined for the identification of peptides in complex mixtures as an alternative to widely used two-dimensional gel electrophoresis followed by MS analysis for use in proteomics. The present method involves the off-line coupling of a narrow-bore, polymer-based, reversed-phase column using an acetonitrile gradient in an alkaline mobile phase in the first dimension with octadecylsilanized silica (ODS)-based nano-LC/MS in the second dimension. After the first separation, successive fractions were acidified and dried off-line, then loaded on the second dimension column. Both columns separate peptides according to hydrophobicity under different pH conditions, but more peptides were identified than with the conventional technique for shotgun proteomics, that is, the combination of a strong cation exchange column with an ODS column, and the system was robust because no salts were included in the mobile phases. The suitability of the method for proteomics measurements was evaluated.     

Label-free quantification in clinical proteomics

Nowadays, proteomic studies no longer focus only on identifying as many proteins as possible in a given sample, but aiming for an accurate quantification of them. Especially in clinical proteomics, the investigation of variable protein expression profiles can yield useful information on pathological pathways or biomarkers and drug targets related to a particular disease. Over the time, many quantitative proteomic approaches have been established allowing researchers in the field of proteomics to refer to a comprehensive toolbox of different methodologies. In this review we will give an overview of different methods of quantitative proteomics with focus on label-free proteomics and its use in clinical proteomics.     

18O-labeling quantitative proteomics using an ion trap mass spectrometer

We describe a method for simultaneous identification and quantitation of proteins within complex mixtures. The method consists of 18O-labeling, a simple stable isotope-coding that requires merely enzymatic digestion in 18O-water, in combination with a capillary-liquid chromatography electrospray ion-trap mass spectrometer. In a separate experiment using the same sample and a spike test, we demonstrate that the difference ration was calculated accurately using the 18O-labeling method even if the protein was part of a complex mixture. Our data also suggest that the accuracy of the quantitation can be improved by averaging the difference ratios of several peptides. In comparing our method with the isotope-coded affinity tag (ICAT) method, we show that the 18O-labeling method has the advantages of better recovery and fewer isotope effects. Therefore, the 18O-labeling method is a powerful tool for large-scale proteomics applications.     

Comparison of DIGE and post-stained gel electrophoresis with both traditional and SameSpots analysis for quantitative proteomics

2-DE is an important tool in quantitative proteomics. Here, we compare the deep purple (DP) system with DIGE using both a traditional and the SameSpots approach to gel analysis. Missing values in the traditional approach were found to be a significant issue for both systems. SameSpots attempts to address the missing value problem. SameSpots was found to increase the proportion of low volume data for DP but not for DIGE. For all the analysis methods applied in this study, the assumptions of parametric tests were met. Analysis of the same images gave significantly lower noise with SameSpots (over traditional) for DP, but no difference for DIGE. We propose that SameSpots gave lower noise with DP due to the stabilisation of the spot area by the common spot outline, but this was not seen with DIGE due to the co-detection process which stabilises the area selected. For studies where measurement of small abundance changes is required, a cost-benefit analysis highlights that DIGE was significantly cheaper regardless of the analysis methods. For studies analysing large changes, DP with SameSpots could be an effective alternative to DIGE but this will be dependent on the biological noise of the system under investigation.     

A novel strategy for quantitative proteomics using isotope-coded protein labels

Stable isotope labelling in combination with mass spectrometry has emerged as a powerful tool to identify and relatively quantify thousands of proteins within complex protein mixtures. Here we describe a novel method, termed isotope-coded protein label (ICPL), which is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on stable isotope tagging at the frequent free amino groups of isolated intact proteins, it is applicable to any protein sample, including extracts from tissues or body fluids, and compatible to all separation methods currently employed in proteome studies. The method showed highly accurate and reproducible quantification of proteins and yielded high sequence coverage, indispensable for the detection of post-translational modifications and protein isoforms. The efficiency (e.g. accuracy, dynamic range, sensitivity, speed) of the approach is demonstrated by comparative analysis of two differentially spiked proteomes.     

Life‐style changes of a halophilic archaeon analyzed by quantitative proteomics

Quantitative proteomics based on isotopic labeling has become the method of choice to accurately determine changes in protein abundance in highly complex mixtures. Isotope‐coded protein labeling (ICPL), which is based on the nicotinoylation of proteins at lysine residues and free N‐termini was used as a simple, reliable and fast method for the comparative analysis of three different cellular states of the halophilic archaeon Halobacterium salinarum through pairwise comparison. The labeled proteins were subjected to SDS‐PAGE, in‐gel digested and the proteolytic peptides were separated by LC and analyzed by MALDI‐TOF/TOF MS. Automated quantitation was performed by comparing the MS peptide signals of ¹²C and ¹³C nicotinoylated isotopic peptide pairs. The transitions between (i) aerobic growth in complex versus synthetic medium and (ii) aerobic versus anaerobic/phototrophic growth, both in complex medium, provide a wide span in nutrient and energy supply for the cell and thus allowed optimal studies of proteome changes. In these two studies, 559 and 643 proteins, respectively, could be quantified allowing a detailed analysis of the adaptation of H. salinarum to changes of its living conditions. The subtle cellular response to a wide variation of nutrient and energy supply demonstrates a fine tuning of the cellular protein inventory.     

Fully automatable two-dimensional reversed-phase capillary liquid chromatography with online tandem mass spectrometry for shotgun proteomics

Herein, we describe the development of a fully automatable technology that features online coupling of high‐pH RP separation with conventional low‐pH RP separation in a two‐dimensional capillary liquid chromatography (2‐D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first‐dimension, high‐pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second‐dimension, low‐pH RP separation, each under identical gradient‐elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non‐redundant proteins, of which 50% were observed in all three replicates. A comparison of RP‐RP 2‐D LC and strong cation exchange‐RP 2‐D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries.     

Global identification of miR-373-regulated genes in breast cancer by quantitative proteomics

Although microRNAs (miRNAs) have been reported to play an important role in carcinogenesis, their molecular mechanism remains largely unknown because of our limited understanding of miRNA target genes. miR-373 was found to be capable of promoting breast cancer invasion and metastasis, but only a target gene was experimentally identified on the basis of mRNA expression analysis. In this study, we used SILAC-based quantitative proteomics to globally identify the genes regulated by miR-373. Totally, 3666 proteins were identified, and 335 proteins were found to be regulated by miR-373. Among the 192 proteins that were downregulated by miR-373, 27 (14.1%) were predicted to have at least one potential match site at their 3'-UTR for miR-373 seed sequence. However, miR-373 did not affect the mRNA level of the five selected candidate targets, TXNIP, TRPS1, RABEP1, GRHL2 and HIP1, suggesting that the protein expressions were regulated by miR-373 via translational inhibition instead of mRNA degradation. Luciferase and mutation assays validated that TXNIP and RABEP1 were the direct target genes of miR-373. More than 30 proteins reported to be involved in cancer invasion and metastasis were found to be regulated by miR-373 in breast cancer for the first time.     

The methods of quantitative proteomics

In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and funciotnal homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.     

Mass spectrometry-based quantitative proteomics

A major aim of present-day proteomics is to study changes in protein expression levels at a global level, ideally monitoring all proteins present in cells or tissue. Mass spectrometry is a well-respected technology in proteomics that is widely used for the identification of proteins. More recently, methodologies have been introduced showing that mass spectrometry can also be used for protein quantification. This article reviews various mass spectrometry-based technologies in quantitative proteomics, highlighting several interesting applications in areas ranging from cell biology to clinical applications.     

Amino acid-coded tagging approaches in quantitative proteomics

To improve the efficiency, accuracy, reproducibility, throughput and proteome coverage of mass spectrometry-based quantitative approaches, both in vitro and in vivo tagging of particular amino acid residues of cellular proteins have been introduced to assist mass spectrometry for global-scale comparative studies of differentially expressed proteins/modifications between different biologically relevant cell states or cells at different pathological states. The basic features of these methods introduce pair-wise isotope signals of each individual peptide containing a particular type of tagged amino acid (amino acid-coded mass tagging) that originated from different cell states. In this review, the applications of major amino acid-coded mass tagging-based quantitative proteomics approaches, including isotope-coded affinity tag, isobaric tags for relative and absolute quantification (iTRAQ?) and stable isotope labeling by amino acids in cell culture are summarized in the context of their respective strengths/weakness in identifying those differentially expressed or post-translational modified proteins regulated by particular cellular stress on a genomic scale in a high-throughput manner. Importantly, these gel-free, in-spectra quantitative mechanisms have been further explored to identify/characterize large-scale protein–protein interactions involving various functional pathways. Taken together, the information about quantitative proteome changes, including multiple regulated proteins and their interconnected relationships, will provide an important insight into the molecular mechanisms, where novel targets for diagnosis and therapeutic intervention will be identified.     

Technical strategies to reduce the amount of "false significant" results in quantitative proteomics

When the p-value is set at <0.05 in statistical group comparisons, a 5% rate of "false significant" results is expected. In order to test the reliability of our 2-DE method, we loaded each of 24 gels with equal-sized samples (200 mug protein from pooled rat brain, pH 4-7, stained with ruthenium fluorescent stain for visualization) and statistically compared the first 12 gels with the last 12. In numerous experiments the rate of significant differences found far exceeded 5%. Several factors were identified as causing the following rates of false significant differences in spot intensities: (i) running samples in two different 2-DE runs (42%), (ii) running second dimension gels produced in two different gel casters (16%), (iii) normalizing the entire gel instead of separately normalizing several different gel zones (11%), (iv) using IPG strips from different packages (19%), (v) dividing the whole sample into subgroups during software analysis (9%). After controlling for all these factors, the rates of "false positive" results in our experiments were regularly reduced to approximately 5%. This is an indispensable prerequisite for avoiding too high a rate of false positive results in experiments in which different subgroups are compared statistically.     

Accurate quantitation of standard peptides used for quantitative proteomics

MS‐based proteomics has become an indispensable tool in system biology generating a need for accurate and precise quantitation of peptide standards. The presented method utilizes ultra performance LC‐MS/MS (UPLC‐MS/MS) to accurately quantify peptide standards at concentrations of 0.1–10 μM. The ability for accurate quantitation of micro‐molar concentrations has the advantages that quantitation can be performed routinely with high precision and the high sensitivity of the method minimizes the amounts required.     

基于质谱技术的蛋白质组定量方法

对不同状态下的蛋白质在表达和修饰水平上进行精确定量,对于探索蛋白质的生物功能、发现疾病的生物标志物都具有重要意义,也是当前蛋白质组学的一个重要研究前沿。近年来,各种蛋白质组定量的新技术和新方法不断涌现,但仍面临着巨大挑战。本文就基于质谱技术的多种蛋白质组定量方法的基本原理、近几年的研究进展和应用进行评述。     

Investigation of ovarian cancer associated sialylation changes in N-linked glycopeptides by quantitative proteomics

ABSTRACT: BACKGROUND: In approximately 80% of patients, ovarian cancer is diagnosed when the patient is already in the advanced stages of the disease. CA125 is currently used as the marker for ovarian cancer; however, it lacks specificity and sensitivity for detecting early stage disease. There is a critical unmet need for sensitive and specific routine screening tests for early diagnosis that can reduce ovarian cancer lethality by reliably detecting the disease at its earliest and treatable stages. Results: In this study, we investigated the N-linked sialylated glycopeptides in serum samples from healthy and ovarian cancer patients using Lectin-directed Tandem Labeling (LTL) and iTRAQ quantitative proteomics methods. We identified 45 N-linked sialylated glycopeptides containing 46 glycosylation sites. Among those, ten sialylated glycopeptides were significantly up-regulated in ovarian cancer patients' serum samples. LC-MS/MS analysis of the non-glycosylated peptides from the same samples, western blot data using lectin enriched glycoproteins of various ovarian cancer type samples, and PNGase F (+/-) treatment confirmed the sialylation changes in the ovarian cancer samples. Conclusion: Herein, we demonstrated that several proteins are aberrantly sialylated in N-linked glycopeptides in ovarian cancer and detection of glycopeptides with abnormal sialylation changes may have the potential to serve as biomarkers for ovarian cancer.     

Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as (15)N. By utilising hydroponic media that contain (15)N inorganic salts as the sole nitrogen source, near to 100% (15)N-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled (14)N- and (15)N-hydroponically grown plants were fractionated by SDS-PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Proteins were identified and the spectra of (14)N/(15)N peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the (14)N and (15)N peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct (14)N and (15)N peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2).