Enhanced information output from shotgun proteomics data by protein quantification and peptide quality control (PQPQ)

We present a tool to improve quantitative accuracy and precision in mass spectrometry based on shotgun proteomics: protein quantification by peptide quality control, PQPQ. The method is based on the assumption that the quantitative pattern of peptides derived from one protein will correlate over several samples. Dissonant patterns arise either from outlier peptides or because of the presence of different protein species. By correlation analysis, protein quantification by peptide quality control identifies and excludes outliers and detects the existence of different protein species. Alternative protein species are then quantified separately. By validating the algorithm on seven data sets related to different cancer studies we show that data processing by protein quantification by peptide quality control improves the information output from shotgun proteomics. Data from two labeling procedures and three different instrumental platforms was included in the evaluation. With this unique method using both peptide sequence data and quantitative data we can improve the quantitative accuracy and precision on the protein level and detect different protein species.     

Method for the simultaneous quantitation of apolipoprotein E isoforms using tandem mass spectrometry

Using apolipoprotein E (ApoE) as a model protein, we developed a protein isoform analysis method utilizing stable isotope labeling tandem mass spectrometry (SILT MS). ApoE isoforms are quantitated using the intensities of the b and y ions of the ¹³C-labeled tryptic isoform-specific peptides versus unlabeled tryptic isoform-specific peptides. The ApoE protein isoform analysis using SILT allows for the simultaneous detection and relative quantitation of different ApoE isoforms from the same sample. This method provides a less biased assessment of ApoE isoforms compared to antibody-dependent methods, and may lead to a better understanding of the biological differences between isoforms.     

Structural proteomics of macromolecular assemblies using oxidative footprinting and mass spectrometry

Understanding the composition, structure and dynamics of macromolecules and their assemblies is at the forefront of biological science today. Hydroxyl-radical-mediated protein footprinting using mass spectrometry can define macromolecular structure, macromolecular assembly and conformational changes of macromolecules in solution based on measurements of reactivity of amino acid side-chain groups with covalent-modification reagents. Subsequent to oxidation by reactive oxygen species, proteins are digested by specific proteases to generate peptides for analysis by mass spectrometry. Accurate measurements of side-chain reactivity are achieved using quantitative liquid-chromatography-coupled mass spectrometry, whereas the side-chain sites within the macromolecular probes are identified using tandem mass spectrometry. In addition, the use of footprinting data in conjunction with computational modeling approaches is a powerful new method for testing and refining structural models of macromolecules and their complexes.     

Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry

The objective of this study was to determine if liquid chromatography mass spectrometry (LC/MS) data of tryptic digests of proteins can be used for quantitation. In theory, the peak area of peptides should correlate to their concentration; hence, the peak areas of peptides from one protein should correlate to the concentration of that particular protein. To evaluate this hypothesis, different amounts of tryptic digests of myoglobin were analyzed by LC/MS in a wide range between 10 fmol and 100 pmol. The results show that the peak areas from liquid chromatography mass spectrometry correlate linearly to the concentration of the protein (r2 = 0.991). The method was further evaluated by adding two different concentrations of horse myoglobin to human serum. The results confirm that the quantitation method can also be used for quantitative profiling of proteins in complex mixtures such as human sera. Expected and calculated protein ratios differ by no more than 16%. We describe a new method combining protein identification with accurate profiling of individual proteins. This approach should provide a widely applicable means to compare global protein expression in biological samples.     

Protein phosphorylation and expression profiling by Yin-yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry

A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange (SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX column. The flow-through peptides from SCX were collected and further loaded on an SAX column. Both columns were eluted by offline pH steps, and the collected fractions were identified by reverse-phase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with 809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134 groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but different phosphorylated states. This unbiased profiling of protein expression and phosphorylation provides a powerful approach to probe protein dynamics, without using any prefractionation and chemical derivation.     

Bioinformatics meets proteomics--bridging the gap between mass spectrometry data analysis and cell biology

Proteomics research programs typically comprise the identification of protein content of any given cell, their isoforms, splice variants, post-translational modifications, interacting partners and higher-order complexes under different conditions. These studies present significant analytical challenges owing to the high proteome complexity and the low abundance of the corresponding proteins, which often requires highly sensitive and resolving techniques. Mass spectrometry plays an important role in proteomics and has become an indispensable tool for molecular and cellular biology. However, the analysis of mass spectrometry data can be a daunting task in view of the complexity of the information to decipher, the accuracy and dynamic range of quantitative analysis, the availability of appropriate bioinformatics software and the overwhelming size of data files. The past ten years have witnessed significant technological advances in mass spectrometry-based proteomics and synergy with bioinformatics is vital to fulfill the expectations of biological discovery programs. We present here the technological capabilities of mass spectrometry and bioinformatics for mining the cellular proteome in the context of discovery programs aimed at trace-level protein identification and expression from microgram amounts of protein extracts from human tissues.     

The selected reaction monitoring/multiple reaction monitoring-based mass spectrometry approach for the accurate quantitation of proteins: clinical applications in the cardiovascular diseases

Selected reaction monitoring, also known as multiple reaction monitoring, is a powerful targeted mass spectrometry approach for a confident quantitation of proteins/peptides in complex biological samples. In recent years, its optimization and application have become pivotal and of great interest in clinical research to derive useful outcomes for patient care. Thus, selected reaction monitoring/multiple reaction monitoring is now used as a highly sensitive and selective method for the evaluation of protein abundances and biomarker verification with potential applications in medical screening. This review describes technical aspects for the development of a robust multiplex assay and discussing its recent applications in cardiovascular proteomics: verification of promising disease candidates to select only the highest quality peptides/proteins for a preclinical validation, as well as quantitation of protein isoforms and post-translational modifications.     

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).     

Accurate mass comparison coupled with two endopeptidases enables identification of protein termini

Protein termini play important roles in biological processes, but there have been few methods for comprehensive terminal proteomics. We have developed a new method that can identify both the amino and the carboxyl termini of proteins. The method independently uses two proteases, (lysyl endopeptidase) Lys-C and peptidyl-Lys metalloendopeptidase (Lys-N), to digest proteins, followed by LC-MS/MS analysis of the two digests. Terminal peptides can be identified by comparing the peptide masses in the two digests as follows: (i) the amino terminal peptide of a protein in Lys-C digest is one lysine residue mass heavier than that in Lys-N digest; (ii) the carboxyl terminal peptide in Lys-N digest is one lysine residue mass heavier than that in Lys-C digest; and (iii) all internal peptides give exactly the same molecular masses in both the Lys-C and the Lys-N digest, although amino acid sequences of Lys-C and Lys-N peptides are different (Lys-C peptides end with lysine, whereas Lys-N peptides begin with lysine). The identification of terminal peptides was further verified by examining their MS/MS spectra to avoid misidentifying pairs as termini. In this study, we investigated the usefulness of this method using several protein and peptide mixtures. Known protein termini were successfully identified. Acetylation on N-terminus and protein isoforms, which have different termini, was also determined. These results demonstrate that our new method can confidently identify terminal peptides in protein mixtures.     

Automated selected reaction monitoring software for accurate label-free protein quantification

Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.     

Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry

Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and β-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633-641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20-30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications.     

Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis.

We report the use of microbore reverse-phase high performance liquid chromatography connected on-line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow-splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer. The method was used to analyze the 265-kDa and the 280-kDa isoforms of the enzyme acetyl-CoA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error-free data interpretation in chemical high-sensitivity peptide sequence analysis.     

Quantification of proteome dynamics in Corynebacterium glutamicum by (15)N-labeling and selected reaction monitoring

Selected reaction monitoring allows quantitative measurements of proteins over several orders of magnitude in complex biological samples. Here we present a targeted approach for quantification of 19 enzymes from Corynebacterium glutamicum applying isotope dilution mass spectrometry coupled to high performance liquid chromatography (IDMS-LC-MS/MS). Investigations of protein dynamics upon growth on acetate and glucose as sole carbon source shows highly stable peptide amounts for enzymes of the central carbon metabolism during the transition phase and after substrate depletion. However significant adaptations of protein amounts are observed between both growth conditions well agreeing with known changes in metabolic fluxes. Time-resolved measurements of protein expression after metabolic switch from glycolytic to gluconeogenetic conditions reveal fast responses in protein synthesis rates for glyoxylate shunt enzymes.     

A recombinant courtship pheromone affects sexual receptivity in a plethodontid salamander

Pheromones are important chemical signals for many vertebrates, particularly during reproductive interactions. In the terrestrial salamander Plethodon shermani, a male delivers proteinaceous pheromones to the female as part of their ritualistic courtship behavior. These pheromones increase the female's receptivity to mating, as shown by a reduction in courtship duration. One pheromone component in particular is plethodontid receptivity factor (PRF), a 22-kDa protein with multiple isoforms. This protein alone can act as a courtship pheromone that causes the female to be more receptive. We used a bacterial expression system to synthesize a single recombinant isoform of PRF. The recombinant protein was identical to the native PRF, based on mass spectrometry, circular dichroism spectra, and a behavioral bioassay that tested the effects of recombinant PRF (rPRF) on female receptivity (21% reduction in courtship duration). The rPRF appears to mimic the activity of a mixture of PRF isoforms, as well as a mixture of multiple different proteins that comprise the male courtship gland extract. Pheromones that are peptides have been characterized for some vertebrates; to date, however, rPRF is one of only 2 synthesized vertebrate proteins to retain full biological activity.     

Elucidating protein inter- and intramolecular interacting domains using chemical cross-linking and matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry

Among many methods used to investigate protein/protein interactions, chemical cross-linking combined with mass spectrometry remains a vital experimental approach. Mapping peptides modified by cross-linker provides clues about proteins’ interacting domains. One complication is that such modification may result from intra- but not intermolecular interactions. Therefore, for overall data interpretation, a combination of results from various platforms is necessary. It is postulated that the secretory isoform of gelsolin regulates several biological processes through interactions with proteins such as actin, fibronectin, vitamin D-binding protein, and unidentified receptors on the surface of eukaryotes; it also has been shown to self-assemble eventually leading to the formation of homo-multimers. As such, it is an excellent model for this study. We used four cross-linkers with arm length ranging from 7.7 to 21.7 Å and MALDI-TOF/TOF mass spectrometry as the analytical platform. Results of this study show that MALDI-based mass spectrometry generates high quality data to show lysine residues modified by cross-linkers and combined with existing data based on crystallography (Protein Data Bank, PDB) can be used to discriminate between inter- and intramolecular linking.