Characterization of the low molecular weight human serum proteome

Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.     

Nanoporous surfaces as harvesting agents for mass spectrometric analysis of peptides in human plasma

Silica-based nanoporous surfaces have been developed in order to capture low molecular weight peptides from human plasma. Harvested peptides were subjected to mass spectrometric analysis by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of detecting and assessing the bound molecules. Peptide profiles consisting of about 70 peaks in the range 800-10,000 m/z were generated. The method could allow detection of small peptides at ng/mL concentration levels, either in standard solutions or in plasma. The same molecular cutoff effect was observed for mixtures of standard proteins and peptides incubated with silicon-based nanoporous surfaces.     

The low molecular weight proteome of Halobacterium salinarum

Systematic investigation of low molecular weight proteins (LMW, below 20 kDa) in the archaeon Halobacterium salinarum resulted in a 6-fold enhancement of the identification rate, reaching 35% of the theoretical proteome in that size range. This was achieved by optimization of common protocols for protein analysis with general applicability. LMW proteins were rapidly and effectively enriched by filter membrane centrifugation followed by tricine SDS-PAGE. Without staining and with significantly shortened digestion protocols, LMW proteins were identified using an FT-ICR mass spectrometer which allows reliable protein identification by MS3 of a single peptide. In addition to a series of technical challenges, small proteins may show low gene expression levels as suggested by their low average codon adaptation index. Twenty functionally uncharacterized proteins contain a characteristic DNA/RNA binding zinc finger motif which underlines the biological relevance of the small proteome and the necessity of their analysis for systems biology.     

Detection and characterization of N-alpha-chloramines by electrospray tandem mass spectrometry

Hypochlorous acid (HOCl) is a major product of activated neutrophils and may be important in antimicrobial activities of cells by oxidation or chlorination of susceptible amino acids. Three major peaks separated using C18 reverse phase-high-performance liquid chromatography RP-HPLC after incubation of leucine enkephalin (LeuEnk) with HOCl. Electrospray mass spectrometry showed masses of m/z 556.2, 590.2, and 624.4 corresponding to unmodified LeuEnk and peptides altered by addition of one or two chlorines (Cl). Formation of stable N-alpha-chloramines was indicated because the chlorinated peptides were readily reduced with the physiological reductants glutathione and ascorbic acid to LeuEnk (m/z 556.2) within 10 min. Sequence-specific ions observed in product ion spectra of single-charged monochlorinated and dichlorinated peptides were consistent with modification of the N-terminal amine. There was no evidence for chlorination of the Tyr aromatic ring in any spectra. Similar RP-HPLC profiles were obtained after oxidation of des-Tyr1-LeuEnk (GGFL) with the masses of the major products being m/z 393.3, 427.2, and 461.1. These were identified as unmodified GGFL, N-alpha-Cl-GGFL, and N-alpha-Cl2-GGFL based on comparison of tandem mass spectra. Oxidation of Met and formation of disulfide dimers was observed after incubation of either N-alpha-Cl-LeuEnk or N-alpha-Cl2-LeuEnk with a protein, indicating that both peptide N-alpha-chloramines were able to readily modify sulfur-containing amino acids within proteins. These data indicate initial formation of stable N-alpha-chorinated peptides after incubation with HOCl and suggest that N-alpha-chlorinated peptides may exist for some hours in the absence of physiological reducing agents or sulfur-containing amino acids.     

Characterization of a diverse secretome generated by the mouse preimplantation embryo in vitro

This study investigates the suitability of surface-enhanced laser desorption and ionization time-of-flight (SELDI-TOF) and electrospray ionization (ESI) mass spectrometry for analysis of the proteins released by the mouse preimplantation embryo in vitro. SELDI-TOF analysis with CM10 or IMAC30 (but not Q10) protein chips detected a protein peak at m/z ~8570 released by both C57BL6 and hybrid embryos. No other peaks unique to the presence of the embryo were identified with this method. ESI mass spectrometry of tryptic digests of embryo-conditioned media identified a total of 20 proteins released during development from the zygote to blastocyst stage. Four proteins were expressed in at least 7 out of 8 cultures tested, one of these (lactate dehydrogenase B) was in all cultures. A further five proteins were in at least half of the cultures and 11 more proteins were in at least one culture. The expression of two of these proteins is essential for preimplantation embryo development (NLR family, pyrin domain containing 5 and peptidyl arginine deiminase, type VI). A further four proteins detected have roles in redox regulation of cells, and three others are capable of inducing post-translational modifications of proteins. This study shows the feasibility of ESI mass spectrometry for identifying the proteins secreted by the preimplantation embryo in vitro. This analysis identifies a range of targets that now require detailed functional analysis to assess whether their release by the embryo is an important property of early embryo development.     

Proteomic investigation of natural killer cell microsomes using gas-phase fractionation by mass spectrometry

We have explored the utility of gas-phase fractionation by mass spectrometry (MS) in the mass-to-charge (m/z) dimension (GPF(m/z)) for increasing the effective number of protein identifications in cases where sample quantity limits the use of multi-dimensional chromatographic fractionation. A peptide digestate from proteins isolated from the membrane fraction of natural killer (NK) cells was analyzed by microcapillary reversed-phase liquid chromatography coupled online to an ion-trap (IT) mass spectrometer. Performing GPF(m/z) using eight narrow precursor ion scan m/z ranges enabled the identification of 340 NK cell proteins from 12 microg of digestate, representing more than a fivefold increase in the number of proteins identified as compared to the same experiment employing a standard precursor ion survey scan m/z range (i.e., m/z 400-2000). The results show that GPF(m/z) represents an effective technique for increasing protein identifications in global proteomic investigations especially when sample quantity is limited.     

A signature-based method to distinguish time-of-flight secondary-ion mass spectra from biological samples

Time-Of-Flight Mass Spectrometry (TOF-SIMS) was used to determine elemental and biomolecular ions from isolated protein samples. We identified a set of 23 mass-to-charge ratio (m/z) peaks that represent signatures for distinguishing biological samples. The 23 peaks were identified by Singular Value Decomposition (SVD) and Canonical Analysis (CA) to find the underlying structure in the complex mass-spectra data sets. From this modified data, SVD was used to identify sets of m/z peaks, and we used these patterns from the TOF-SIMS data to predict the biological source from which individual mass spectra were generated. The signatures were validated using an additional data set different from the initial training set used to identify the signatures. We present a simple method to identify multiple variables required for sample classification based on mass spectra that avoids overfit. This is important in a variety of studies using mass spectrometry, including the ability to identify proteins in complex mixtures and for the identification of new biomarkers.     

The amplified peptidome: the new treasure chest of candidate biomarkers

Mass spectrometric analysis of the low-molecular weight (LMW) range of the serum/plasma proteome is revealing the existence of large numbers of previously unknown peptides and protein fragments predicted to be derived from low-abundance proteins. This raises the question of why such low abundance molecules would be retained at detectable levels in the circulation, instead of being rapidly cleared and excreted. Theoretical models of biomarker production and association with serum carrier proteins have been developed to elucidate the mechanisms governing biomarker half-life in the bloodstream. These models predict that the vast majority of LMW biomarkers exist in association with circulating high molecular mass carrier proteins. Moreover, the total serum/plasma concentration of the biomarker is largely determined by the clearance rate of the carrier protein, not the free-phase biomarker clearance itself. These predictions have been verified experimentally using molecular mass fractionation of human serum before mass spectrometry sequence analysis. These principles have profound implications for biomarker discovery and measurement.     

Regulation of protein synthesis in hen's oviducts. II. Extracellular ovalbumin as an inhibitor of ovalbumin synthesis in the oviducts.

Washed, preincubated minced hen's oviducts, which contained low levels of extracellular and intracellular proteins, synthesized egg-white proteins actively. The addition of ovalbumin to the incubation medium resulted in inhibition of the synthesis of egg-white proteins by the washed, preincubated oviduct cells, while the addition of bovine serum albumin seemed to stimulate protein synthesis and hen's egg-white lysozyme had no effect. The inhibitory or stimulatory effect on protein synthesis was proportional to the amount of protein added to the medium. The inhibitory effect of added ovalbumin was shown not to be due to the incorporation of ovalbumin into the oviduct cells from the incubation medium. Egg-white proteins added to the medium also inhibited protein synthesis inside the cells and the extent of the inhibition appeared to correspond to the amount of ovalbumin present in egg-white.     

Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.     

Biomarkers of HIV-1 associated dementia: proteomic investigation of sera

Background

Acute lymphoblastic leukemia (ALL) is a common form of cancer in children. Currently, bone marrow biopsy is used for diagnosis. Noninvasive biomarkers for the early diagnosis of pediatric ALL are urgently needed. The aim of this study was to discover potential protein biomarkers for pediatric ALL.

Methods

Ninety-four pediatric ALL patients and 84 controls were randomly divided into a "training" set (45 ALL patients, 34 healthy controls) and a test set (49 ALL patients, 30 healthy controls and 30 pediatric acute myeloid leukemia (AML) patients). Serum proteomic profiles were measured using surface-enhanced laser desorption/ionization-time-of-flight mass spectroscopy (SELDI-TOF-MS). A classification model was established by Biomarker Pattern Software (BPS). Candidate protein biomarkers were purified by HPLC, identified by LC-MS/MS and validated using ProteinChip immunoassays.

Results

A total of 7 protein peaks (9290 m/z, 7769 m/z, 15110 m/z, 7564 m/z, 4469 m/z, 8937 m/z, 8137 m/z) were found with differential expression levels in the sera of pediatric ALL patients and controls using SELDI-TOF-MS and then analyzed by BPS to construct a classification model in the "training" set. The sensitivity and specificity of the model were found to be 91.8%, and 90.0%, respectively, in the test set. Two candidate protein peaks (7769 and 9290 m/z) were found to be down-regulated in ALL patients, where these were identified as platelet factor 4 (PF4) and pro-platelet basic protein precursor (PBP). Two other candidate protein peaks (8137 and 8937 m/z) were found up-regulated in the sera of ALL patients, and these were identified as fragments of the complement component 3a (C3a).

Conclusion

Platelet factor (PF4), connective tissue activating peptide III (CTAP-III) and two fragments of C3a may be potential protein biomarkers of pediatric ALL and used to distinguish pediatric ALL patients from healthy controls and pediatric AML patients. Further studies with additional populations or using pre-diagnostic sera are needed to confirm the importance of these findings as diagnostic markers of pediatric ALL.     

Proteomic identification of haptoglobin as a stroke plasma biomarker in spontaneously hypertensive stroke-prone rats

AIMS: We investigated changes in the expression of plasma proteins in spontaneously hypertensive stroke-prone rats (SHRSP) to identify stroke biomarkers. MAIN METHODS AND KEY FINDINGS: The present analysis using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) demonstrated that three peaks at mass/charge ratios (m/z) of 9330, 9480 and 9700 decreased in intensity during the development and progression of hypertensive stroke in SHRSPs, but not in age-matched control SHR and Wistar rats. Administration of verapamil, an L-type calcium channel blocker which was effective for hypertension in SHRSP rats, prevented the decrease in plasma protein expression. A candidate biomarker protein (m/z 9330) was identified using LC-MS/MS as haptoglobin (Hp). Immunoblotting with anti-Hp antibody demonstrated the decreased expression of both Hpalpha and Hpbeta chains in SHRSP. In contrast, haptoglobin mRNA expression in the liver of SHRSPs slightly increased as compared with control rats. SIGNIFICANCE: These findings suggest that Hp is a biomarker candidate for discriminating pathogenic alterations of stroke.     

Failure to verify the presence of pyrroloquinoline quinone (PQQ) in bovine plasma amine oxidase by gas chromatography/mass spectrometry

The presence of covalently bound pyrroloquinoline quinone (PQQ) in bovine plasma amine oxidase (BPAO) was examined by the use of gas chromatography/mass spectrometry. The enzyme was subjected to proteolysis with proteinase in the presence of [U-13C]PQQ as an internal standard. After isolation and derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ, respectively, by selected ion monitoring (SIM). In the SIM profile, although the sample extract obtained from BPAO treated with proteinase clearly showed the peak at m/z 462 for the internal standard, there were no peaks detectable at m/z 448, showing the absence of PQQ in the proteolysis digest of BPAO. Thus, our results do not support the claim that BPAO contains covalently bound PQQ in its structure.     

Development of Gel-Filter Method for High Enrichment of Low-Molecular Weight Proteins from Serum

The human serum proteome has been extensively screened for biomarkers. However, the large dynamic range of protein concentrations in serum and the presence of highly abundant and large molecular weight proteins, make identification and detection changes in the amount of low-molecular weight proteins (LMW, molecular weight ≤ 30kDa) difficult. Here, we developed a gel-filter method including four layers of different concentration of tricine SDS-PAGE-based gels to block high-molecular weight proteins and enrich LMW proteins. By utilizing this method, we identified 1,576 proteins (n = 2) from 10 μL serum. Among them, 559 (n = 2) proteins belonged to LMW proteins. Furthermore, this gel-filter method could identify 67.4% and 39.8% more LMW proteins than that in representative methods of glycine SDS-PAGE and optimized-DS, respectively. By utilizing SILAC-AQUA approach with labeled recombinant protein as internal standard, the recovery rate for GST spiked in serum during the treatment of gel-filter, optimized-DS, and ProteoMiner was 33.1 ± 0.01%, 18.7 ± 0.01% and 9.6 ± 0.03%, respectively. These results demonstrate that the gel-filter method offers a rapid, highly reproducible and efficient approach for screening biomarkers from serum through proteomic analyses.     

Phospholipid molecular species distribution of some medically important Candida species analysed by fast atom bombardment mass spectroscopy

The aim of this study was to obtain detailed information on phospholipids (PL) of the medically important Candida species and to determine their possible chemotaxonomic significance. Lipids were extracted from 22 strains representing 8 Candida species and their PL molecular species distributions were determined by Fast Atom Bombardment Mass Spectroscopy (FAB MS) in negative ion mode. Fifteen major lower mass peaks (m/z 221 to 289) were attributable to the expected presence of carboxylate anions and 24 major higher mass peaks (m/z 557 to 837) were attributable to phospholipid anions. Major carboxylate peaks were of the following m/z and identities : 253, C16:1; 255, C16:0; 277, C18:3; 279, C18:2; 281, C18:1; and 283, C18:0. The most abundant peaks consistent with the presence of phospholipid molecular species anions include those of m/z 673, 743, 833, 834 and 836 tentatively identified as phosphatidic acid (PA) (34:1), phosphatidylglycerol (PG) (34:3), phosphtidylinositol (PI) (34:2) and two unknown molecular species. This profile is diagnostic for the genus Candida. Quantitative differences were observed between different Candida species. Thus, polar lipid molecular species distribution in Candida spp. has chemotaxonomic significance, especially so in the case of carboxylate anions.     

Machine learning approaches to lung cancer prediction from mass spectra

We addressed the problem of discriminating between 24 diseased and 17 healthy specimens on the basis of protein mass spectra. To prepare the data, we performed mass to charge ratio (m/z) normalization, baseline elimination, and conversion of absolute peak height measures to height ratios. After preprocessing, the major difficulty encountered was the extremely large number of variables (1676 m/z values) versus the number of examples (41). Dimensionality reduction was treated as an integral part of the classification process; variable selection was coupled with model construction in a single ten-fold cross-validation loop. We explored different experimental setups involving two peak height representations, two variable selection methods, and six induction algorithms, all on both the original 1676-mass data set and on a prescreened 124-mass data set. Highest predictive accuracies (1-2 off-sample misclassifications) were achieved by a multilayer perceptron and Na?ve Bayes, with the latter displaying more consistent performance (hence greater reliability) over varying experimental conditions. We attempted to identify the most discriminant peaks (proteins) on the basis of scores assigned by the two variable selection methods and by neural network based sensitivity analysis. These three scoring schemes consistently ranked four peaks as the most relevant discriminators: 11683, 1403, 17350 and 66107.     

Clarification of mechanism of human sputum elastase inhibition by a new inhibitor,ONO-5046, using electrospray ionization mass spectrometry

Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) to probe the nature of the covalent E-I complex was successfully applied to clarify the mechanism of human sputum elastase (HSE) inhibition by a new inhibitor, ONO-5046. The mass spectrum of the four HSE isozymes displayed their molecular ion peaks at m/z=26,018, 25,929, 25,200, and 25,054, respectively. Immediately after incubation, inactivation of HSE with ONO-5046 increased the four molecular ion peaks by approximately 84 amu, which was assigned to the mass unit of the pivaloyl moiety of ONO-5046. An additional minute of incubation of E-I complex restored the original molecular ion peaks. These observations strongly suggested that ONO-5046 inactivates HSE by a reversible 'acylation-deacylation' mechanism.     

Polar lipids of Staphylococcus strains analysed by fast atom bombardment mass spectrometry

This study used fast atom bombardment mass spectrometry (FAB MS) to obtain detailed information on polar lipids of Staphylococcus by examining 23 isolates. Eighteen major anions were found in the range m/z 199–297, consistent with the presence of carboxylate anions. A further 21 major anions were found in the higher mass regions of m/z 609–805, consistent with the presence of phospholipid anions. In Staph. aureus, Staph. epidermidis, Staph. haemolyticus and Staph. hominis , the most intense peaks putatively assigned as carboxylate ions were consistent with presence of C_15:0, followed by C_17:0 except in the case of Staph. epidermidis. The major phospholipid anions were consistent with the presence of PG(30 : 0), PG(32 : 0) and PG(33 : 0). It is concluded that Staphylococcus has a characteristic polar lipid profile and that qualitative and quantitative differences may be seen between Staph. aureus and Staph. epidermidis.     

Evaluation of matrix-assisted laser desorption/ionization-time of flight mass spectrometry proteomic profiling: identification of alpha 2-HS glycoprotein B-chain as a biomarker of diet

Biomarkers have the potential to impact a wide range of public health concerns, including early detection of diseases, drug discovery, and improved accuracy of monitoring effects of interventions. Given new technological developments, broad-based screening approaches will likely advance biomarker discovery at an accelerated pace. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) allows for the elucidation of individual protein masses from a complex mixture with high throughput. We have developed a method for identifying serum biomarkers using MALDI-TOF and statistical analysis. However, before applying this approach to screening of complex diseases, we evaluated the approach in a controlled dietary intervention study. In this study, MALDI-TOF spectra were generated using samples from a randomized controlled trial. During separate feeding periods, 38 participants ate a basal diet devoid of fruits and vegetables and a basal diet supplemented with cruciferous (broccoli) family vegetables. Serum samples were obtained at the end of each 7-day feeding period and treated to remove large, abundant proteins. MALDI-TOF spectra were analyzed using peak picking algorithms and logistic regression models. Our bioinformatics methods identified two significant peaks at m/z values of 2740 and 1847 that could classify participants based on diet (basal vs. cruciferous) with 76% accuracy. The 2740 m/z peak was identified as the B-chain of alpha 2-HS glycoprotein, a serum protein previously found to vary with diet and be involved in insulin resistance and immune function.     

Rapid Characterization of Microalgae and Microalgae Mixtures Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)

Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of this study were to determine whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels and 2) characterize simple microalgal mixtures. A common protein extraction sample preparation method was used to facilitate rapid mass spectrometry-based analysis of 31 microalgae. Each yielded spectra containing between 6 and 56 peaks in the m/z 2,000 to 20,000 range. The taxonomic resolution of this approach appeared higher than that of 18S rDNA sequence analysis. For example, two strains of Scenedesmus acutus differed only by two 18S rDNA nucleotides, but yielded distinct MALDI-TOF mass spectra. Mixtures of two and three microalgae yielded relatively complex spectra that contained peaks associated with members of each mixture. Interestingly, though, mixture-specific peaks were observed at m/z 11,048 and 11,230. Our results suggest that MALDI-TOF MS affords rapid characterization of individual microalgae and simple microalgal mixtures.