77Se Enrichment of Proteins Expands the Biological NMR Toolbox

Sulfur, a key contributor to biological reactivity, is not amendable to investigations by biological NMR spectroscopy. To utilize selenium as a surrogate, we have developed a generally applicable ⁷⁷Se isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate ⁷⁷Se NMR spectroscopy of multiple selenocysteine and selenomethionine residues in the sulfhydryl oxidase augmenter of liver regeneration (ALR). The resonances of the active-site residues were assigned by comparing the NMR spectra of ALR bound to oxidized and reduced flavin adenine dinucleotide. An additional resonance appears only in the presence of the reducing agent and disappears readily upon exposure to air and subsequent reoxidation of the flavin. Hence, ⁷⁷Se NMR spectroscopy can be used to report the local electronic environment of reactive and structural sulfur sites, as well as changes taking place in those locations during catalysis.     

77Se NMR characterization of 77Se-labeled ovine erythrocyte glutathione peroxidase

Lambs, maintained on a selenium-deficient diet supplemented with 94 atom % Na2 27SeO3, have been used as a source of 77Se-enriched erythrocyte glutathione peroxidase. After 5 months on this diet, the percentage of selenium in the enzyme derived from the supplement had reached 88%. From each monthly bleeding of two sheep, approximately 20 mg of 77Se-enriched glutathione peroxidase could be isolated in pure form. Although attempts to observe 77Se NMR signals from the native enzyme labeled with 6,6'-[77Se]diselenobis-(3-nitrobenzoic acid) failed, due to the low solubility of the enzyme, two 77Se resonances were observed after unfolding the enzyme with 8 M urea and reaction with iodoacetamide. These resonances, at 195 and 377 ppm, were from the selenoether alkylamide derivative and from protein cross-linked selenide sulfide species, respectively. Relaxation time measurements on the selenoether at 4.7 and 9.4 teslas enabled an estimate of the chemical shift anisotropy to be made. A value of less than or equal to 262 ppm was determined. Reduction of the denatured selenide sulfide species with dithiothreitol gave an observable 77Se resonance from the Se- moiety at pH 8 and from SeH at pH 4.2. The chemical form of the selenocysteine residue in the resting state enzyme most consistent with formation of the acetamide derivative and the selenide sulfide is Se- or SeH. From the magnitudes of the estimated chemical shift anisotropies, it is predicted that direct observation of selenium in the native enzyme will be feasible if the enzyme concentration can be increased to 0.25 mM tetrameric glutathione peroxidase.     

Selenium speciation profiles in selenite-enriched soybean (Glycine Max) by HPLC-ICPMS and ESI-ITMS

Soybean (Glycine Max) plants were grown in soil supplemented with sodium selenite. A comprehensive selenium profile, including total selenium concentration, distribution of high molecular weight selenium and characterization of low molecular weight selenium compounds, is reported for each plant compartment: bean, pod, leaf and root of the Se-enriched soybean plants. Two chromatographic techniques, coupled with inductively coupled plasma mass spectrometry (ICPMS) for specific selenium detection, were employed in this work to analyze extract solutions from the plant compartments. Size-exclusion chromatography revealed that the bean compartment, well-known for its strong ability to make proteins, produced high amounts (82% of total Se) of high molecular weight selenospecies, which may offer additional nutritional value and suggest high potential for studying proteins containing selenium in plants. The pod, leaf and root compartments primarily accumulate low molecular weight selenium species. For each compartment, low molecular weight selenium species (lower than 5 kDa) were characterized by ion-pairing reversed phase HPLC-ICPMS and confirmed by electrospray ionization ion trap mass spectrometry (ESI-ITMS). Selenomethionine and selenocystine are the predominant low molecular weight selenium compounds found in the bean, while inorganic selenium was the major species detected in other plant compartments.     

77Se-13C based dipolar correlation experiments to map selenium sites in microcrystalline proteins

Sulfur-containing sites in proteins are of great importance for both protein structure and function, including enzymatic catalysis, signaling pathways, and recognition of ligands and protein partners. Selenium-77 is an NMR active spin-1/2 nucleus that shares many physiochemical properties with sulfur and can be readily introduced into proteins at sulfur sites without significant perturbations to the protein structure. The sulfur-containing amino acid methionine is commonly found at protein–protein or protein–ligand binding sites. Its selenium-containing counterpart, selenomethionine, has a broad chemical shift dispersion useful for NMR-based studies of complex systems. Methods such as (¹H)-⁷⁷Se-¹³C double cross polarization or {⁷⁷Se}-¹³C REDOR could be valuable to map the local environment around selenium sites in proteins but have not been demonstrated to date. In this work, we explore these dipolar transfer mechanisms for structural characterization of the GB1 V39SeM variant of the model protein GB1 and demonstrate that ⁷⁷Se-¹³C based correlations can be used to map the local environment around selenium sites in proteins. We have found that the general detection limit is?~?5 Å, but longer range distances up to?~?7 Å can be observed as well. This study establishes a framework for the future characterization of selenium sites at protein–protein or protein–ligand binding interfaces.

     

NMR relaxation properties of 77Se-labeled proteins

A 77Se-containing moiety has been attached to cysteine residues in bovine hemoglobin, reduced ribonuclease A, and glutathione by reaction with [77Se]6,6'-diselenobis(3-nitrobenzoic acid). The resultant species contain Se-S linkages that have 77Se NMR absorptions in the range range of 568-580 ppm. Spectra have been recorded at 4.7 and 9.7 tesla (T). For labeled hemoglobin a line width of 250 Hz is seen at 4.7 T and 1000 Hz at 9.4 T. This quadrupling of line width with doubling of observational field strength is consistent with exclusive relaxation by the chemical shift anisotropy (CSA) mechanism. These line widths are greater than expected for a molecule the size of hemoglobin and indicate some aggregation at the high concentrations used. Upon dissociation and partial unfolding of the hemoglobin subunits, the line widths of the selenium resonance decrease to 35 and 120 Hz at 4.7 and 9.4 T, respectively. The spin-lattice relaxation time (T1) for the dissociated hemoglobin at 9.4 T was found to be 220 ms. Together with a value of 377 ms for the spin-spin relaxation time (T2), determined from the line width, an estimate of the CSA was made. This gave a value of 890 ppm, which is in accord with other values for Se(II) linked only by single bonds. When this value for the CSA is used, together with the CSA contribution to the line width, in estimating a correlation time for seleno(3-nitrobenzoic acid) (SeNB)-labeled glutathione, a value of 4 x 10(-11) s is obtained. For SeNB-labeled denatured ribonuclease, four distinct resonances are resolvable at 4.7 T and five resonances at 9.4 T. From T1 values for these resonances and the value of 890 ppm for the CSA, an appropriate correlation time of 0.1 ns was determined, which should result in 77Se resonances of 0.2-1.0 Hz at 4.7 and 9.4 T, respectively. Much greater apparent line widths are observed, which are attributed to microheterogeneity resulting from formation of inter- and intramolecular disulfide linkages. It is concluded that when there are no complications from protein aggregation or chemical exchange, the CSA values anticipated to exist in glutathione peroxidase or other selenoproteins should result in resonances with line widths in the range from 27 to 170 Hz, depending on field strength. These resonances should therefore be observable in the intact protein, if 77Se-enriched material is available.     

汞与硒在不同汞暴露水平地区猪肝脏和肾脏蛋白质中的分布

通过对贵州万山汞污染地区及北京地区猪肝脏和肾脏组织上清液进行凝胶过滤色谱分离(SephadexG 10 0 ) ,随后用原子荧光法测定它们蛋白质组分中汞和硒的含量 ,研究在汞暴露水平不同状态下微量元素汞和硒在动物体蛋白质分子水平上的分布 .发现这两个地区猪肝脏和肾脏组织上清液蛋白质组分中汞和硒的分布模式有明显差异 .贵州万山汞污染地区猪肝脏上清液中汞浓度比北京地区高 ,硒浓度也相应高 ,且前者与高分子量和低分子量蛋白结合的硒均明显高于后者 ;而北京地区猪肝脏上清液中的硒主要以与高分子量蛋白结合的形式存在 .贵州汞污染地区猪肝脏上清液中汞主要与高分子量蛋白结合 ,而北京地区猪肝脏上清液中汞则分布较为均匀 .贵州万山地区猪肾脏上清液中 ,含硒峰在高分子量蛋白区和低分子量区都有分布 ;而北京地区猪肾脏上清液中 ,硒则主要集中分布于高分子量蛋白范围 .这两个地区猪肾脏上清液中都有分子量约为 11kD的金属硫蛋白 (MT)存在 ,北京地区猪肾脏上清液中汞主要以与金属硫蛋白结合的形式出现 ,而贵州万山地区猪肾脏上清液中的汞除与金属硫蛋白结合外 ,尚有相当大部分是以与高分子量蛋白结合的形式存在 .研究结果表明 ,由于这两个地区汞暴露水平的差异 ,不仅使这两地区猪肝、肾上清液中的汞与硒含量     

人肝脏组织亚细胞组分中含硒蛋白的分离与测定

硒是生物必需的微量元素 ,具有重要的生物化学功能 ,与人类和动物的健康及疾病密切相关[1,2 ] .生物体内 80 %以上的硒是以与蛋白质结合的形式存在 ,所以目前大量的研究集中在硒蛋白的分离、鉴定和功能研究等方面[3 ] .从 1998年起 ,我们对正常人肝组织中硒和含硒蛋白的亚细胞分布作了一些探索性的工作 ,发现含硒蛋白在各亚细胞组分中的分布有显著的不同[4 ,5] .前期工作采用的凝胶过滤柱层析法研究含硒蛋白 ,该法分离量大 ,但分辨率较差 ,分子量相近的蛋白质难以区分[4 ,5] .本工作采用ＳＤＳ 聚丙烯酰胺凝胶电泳 (ＳＤＳ ＰＡＧＥ)分离人…     

亚硒酸钠诱导的晶状体蛋白质聚合作用

不仅在体内,而且在体外亚硒酸钠可引起晶状体蛋白质聚合。将亚硒酸钠加到pH7.4的晶状体蛋白质溶液中,在37℃保温30min后观察到蛋白质溶液变混浊,随时间的延长混浊程度加重并有沉淀形成。经SDS聚丙烯酰胺凝胶电泳发现,加硒保温后形成的不溶性蛋白质中有大量的高分子聚合物。当加入二硫苏糖醇后混浊的蛋白质溶液变清,其中的高分子聚合物也基本消失,我们还发现;在亚硒酸钠使晶状体蛋白质变混浊的同时,蛋白质巯基减少,而蛋白质结合的硒量增加,且二者之间有较固定的比例关系,即蛋白质上每增加一个硒原子,蛋白质巯基就减少4.26个。当用二硫苏糖醇还原后,68％的硒从蛋白质中释放出来。这些结果表明,亚硒酸钠可引起大鼠晶状体水溶性蛋白质聚合,其可能方式如下:4PSH+SeO_3~-→PSSP+PS-Se-SP+H_2O+2OH~-这可能是亚硒酸钠诱发白内障的主要原因。     

Selenocysteine in proteins-properties and biotechnological use

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK(a) of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.     

A selenium-containing hydrogenase from Methanococcus vannielii. Identification of the selenium moiety as a selenocysteine residue

A 75Se-labeled hydrogenase was purified to near homogeneity from extracts of Methanococcus vannielii cells grown in the presence of [75Se]selenite. The molecular weight of the enzyme was estimated as 340,000 by gel filtration. The enzyme tends to aggregate and occurs also as a larger protein species (Mr = 1.3 x 10(6)). The same phenomenon was observed on native gel electrophoretic analysis. Hydrogenase activity exhibited by these two protein bands was proportional to protein and 75Se content. Both molecular species reduce the natural cofactor, 8-hydroxy-5-deazaflavin, and tetrazolium dyes with molecular hydrogen. Sodium dodecyl sulfate-gel electrophoresis of 75Se-labeled enzyme showed that 75Se is present exclusively in an Mr = 42,000 subunit. A value of 3.8 g atoms of selenium/mol of enzyme (Mr = 340,000) was determined by atomic absorption analysis. The chemical form of selenium in the enzyme was shown to be selenocysteine. This was identified as the [75Se]carboxymethyl and [75Se]carboxyethyl derivatives in acid hydrolysates of alkylated 75Se-labeled protein. The hydrogenase is extremely oxygen-sensitive but can be reactivated by incubation with molecular hydrogen and dithiothreitol.     

Different selenium-containing proteins in the extracellular and intracellular media of leucocytes cultivated in vitro

The purpose of this communication is to elucidate if selenium plays a role in the function of granulocytes and lymphocytes. Thus, the incorpo ration of selenium in proteins from granulocytes and lymphocytes cultured with 1ΜCi/mL radioactive Na₂ ⁷⁵SeO₃ was studied. The protein peaks containing⁷⁵Se from two columns of Heparin Sepharose CL-6B and Sephacryl S-200 HR were separated further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results showed that the incorporation of⁷⁵Se into granulocytes was about six times higher than that of lymphocytes during a 96-h cultivation, however, the GSH-Px activity in granulocytes did not change significantly. On the other hand, the GSH-Px activity of lymphocytes rose significantly after three days cultivation. These data indicated that the main chemical form of selenium in granulocytes was not GSH-Px. Results from SDS-PAGE revealed a strongly⁷⁵Se-labeled protein band with subunit molecular weight of 15 kDa in the supernatant of granulocyte homogenate. However, the main chemical forms of selenium in the culture media of granulocytes and lymphocytes were found to be selenoprotein P. The different forms of selenium-containing proteins in the intracellular and extracellular media of granulocytes indicated the different functions of these proteins.     

A unique selenoprotein isolated from yellowfin tuna (Thunnus albacares) liver

A selenoprotein, with an approximate molecular weight of 2000, was isolated from yellowfin tuna (Thunnus albacares) liver. The selenium (Se) content of this selenoprotein fraction represented greater than 50% of the Se in the original liver extract. Most of the unrecovered Se was left in the pellet following homogenization. Although the protein was very sensitive to oxidizing conditions, it remained stable in the presence of reducing agents such as glutathione and dithiothreitol under a nitrogen atmosphere. After preparative isoelectric focusing of the purified selenoprotein, selenium was detected in three distinct bands, with the predominant band occurring at pH 6.2.     

Role of sulfhydryl compounds in the control of tyrosinase activity in Neurospora crassa

It is known that Neurospora crassa mycelia cultured in standard concentrations (76 to 190 µg/ml) of sulfate accumulate a low molecular weight inhibitor of tyrosinase (monophenol, dihydroxyphenylalanine: oxygen oxidorenductase; EC 1.14.1.18.1.). This is not observed in cultures grown under sulfate-limiting conditions. The chemical nature of tyrosinase inhibition was investigated. It was shown to be due to the low molecular weight sulfhydryl fraction of the extracts, in which glutathione is predominant. The concentration of low molecular weight sulfhydryl compounds decreased sharply in mycelia submitted to various treatments which also derepressed tyrosinase, such as (i) starvation in phosphate buffer, (ii) treatment with cycloheximide, and (iii) mating. These results suggest that the concentration of sulfhydryl compounds may be of physiological significance in the control of tyrosinase activity in N. crassa.     

Choice of Force Field for Proteins Containing Structured and Intrinsically Disordered Regions

Biomolecular force fields optimized for globular proteins fail to properly reproduce properties of intrinsically disordered proteins. In particular, parameters of the water model need to be modified to improve applicability of the force fields to both ordered and disordered proteins. Here, we compared performance of force fields recommended for intrinsically disordered proteins in molecular dynamics simulations of three proteins differing in the content of ordered and disordered regions (two proteins consisting of a well-structured domain and of a disordered region with and without a transient helical motif and one disordered protein containing a region of increased helical propensity). The obtained molecular dynamics trajectories were used to predict measurable parameters, including radii of gyration of the proteins and chemical shifts, residual dipolar couplings, paramagnetic relaxation enhancement, and NMR relaxation data of their individual residues. The predicted quantities were compared with experimental data obtained within this study or published previously. The results showed that the NMR relaxation parameters, rarely used for benchmarking, are particularly sensitive to the choice of force-field parameters, especially those defining the water model. Interestingly, the TIP3P water model, leading to an artificial structural collapse, also resulted in unrealistic relaxation properties. The TIP4P-D water model, combined with three biomolecular force-field parameters for the protein part, significantly improved reliability of the simulations. Additional analysis revealed only one particular force field capable of retaining the transient helical motif observed in NMR experiments. The benchmarking protocol used in our study, being more sensitive to imperfections than the commonly used tests, is well suited to evaluate the performance of newly developed force fields.     

Parameters of dietary selenium and vitamin E deficiency in growing rabbits.

4 x 5 growing female rabbits (New Zealand White) with an initial live weight of 610 +/- 62 g were fed a torula yeast based semisynthetic diet low in selenium (<0.03 mg/kg diet) and containing <2 mg alpha-tocopherol per kg (group I). Group II received a vitamin E supplementation of 150 mg alpha-tocopherylacetate per kg diet, whereas for group III 0.40 mg Se as Na-selenite and for group IV both supplements were added. Selenium status and parameters of tissue damage were analyzed after 10 weeks on experiment (live weight 2,355 +/- 145 g). Selenium depletion of the Se deficient rabbits (groups I and II) was indicated by a significantly lower plasma Se content (group I: 38.3 +/- 6.23 microg Se/mL plasma, group II: 42.6 +/- 9.77, group III: 149 +/- 33.4, group IV: 126 +/- 6.45) and a significantly lower liver Se content (group I: 89.4 +/- 18.2 microg/kg fresh matter, group II: 111 +/- 26.2) as compared to the Se supplemented groups III (983 +/- 204) and IV (926 +/- 73.9). After 5 weeks on the experimental diets differences in the development of plasma glutathione peroxidase were observed. As compared to the initial status group (45.2 +/- 4.50) pGPx activity in mU/mg protein was decreased in group I (19.1 +/- 7.08), remained almost stable in the vitamin E supplemented group II (46.3 +/- 11.2) whereas an elevated enzyme activity was measured in the Se supplemented groups III (62.4 +/- 23.9) and IV (106 +/- 19.9). In the rabbit organs investigated 10 weeks of Se deficiency caused a significant loss of Se dependent cellular glutathione peroxidase activity (GPx1) of 94% (liver), 80% (kidney), 50% (heart muscle) and 60% (musculus longissimus dorsi) in comparison to Se supplemented control animals. Damage of cellular lipids and proteins in the liver was due to either Se or vitamin E deficiency. However damage was most severe under conditions of a combined Se and vitamin E deficiency. It can be concluded that the activity of plasma glutathione peroxidase is a sensitive indicator of Se deficiency in rabbits. The loss of GPx1 activity indicates the selenium depletion in various rabbit organs. Both selenium and vitamin E are essential and highly efficient antioxidants which protect rabbits against lipid and protein oxidation.     

Directly observed 15N NMR spectra of uniformly enriched proteins

The proteins cytochrome c2, cytochrome c', and ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum were enriched in 15N by growth of the organism on 15NH4Cl. The proteins were purified to homogeneity and studied by 15N NMR. Longitudinal and transverse relaxation times as well as the nuclear Overhauser effects were determined for various groups of the proteins which vary in molecular weight from 13,000 to 114,000. The values of these parameters for the amide resonances or for groups thought to be rigid were consistent with the molecular weights of the proteins. Relaxation times of the amino-terminal alpha-amino groups and the side chain nitrogen atoms of arginine and lysine were consistent with much more rapid motion. Nitrogen atoms having bound protons were generally found to be decoupled from the protons by chemical exchange. Demonstrable 1H-15N coupling was taken as an indication that exchange was hindered, either by hydrogen bonding interactions or by inaccessibility of the group to solvent. Histidine side chain nitrogen atoms, which experience a large chemical shift upon protonation/deprotonation, were often found to be broadened beyond detectability by chemical exchange and tautomerization. Strategies for improving sensitivity and for obtaining specific peak assignments are also discussed.     

CP-MAS and Solution NMR Studies of Allosteric Communication in CA-assemblies of HIV-1

Solution and solid-state NMR spectroscopy are highly complementary techniques for studying structure and dynamics in very high molecular weight systems. Here we have analysed the dynamics of HIV-1 capsid (CA) assemblies in presence of the cofactors IP6 and ATPγS and the host-factor CPSF6 using a combination of solution state and cross polarisation magic angle spinning (CP-MAS) solid-state NMR. In particular, dynamical effects on ns to µs and µs to ms timescales are observed revealing diverse motions in assembled CA.Using CP-MAS NMR, we exploited the sensitivity of the amide/Cα-Cβ backbone chemical shifts in DARR and NCA spectra to observe the plasticity of the HIV-1 CA tubular assemblies and also map the binding of cofactors and the dynamics of cofactor-CA complexes. In solution, we measured how the addition of host- and co-factors to CA -hexamers perturbed the chemical shifts and relaxation properties of CA-Ile and -Met methyl groups using transverse-relaxation-optimized NMR spectroscopy to exploit the sensitivity of methyl groups as probes in high-molecular weight proteins. These data show how dynamics of the CA protein assembly over a range of spatial and temporal scales play a critical role in CA function. Moreover, we show that binding of IP6, ATPγS and CPSF6 results in local chemical shift as well as dynamic changes for a significant, contiguous portion of CA, highlighting how allosteric pathways communicate ligand interactions between adjacent CA protomers.     

Selenocysteine in proteins—properties and biotechnological use

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK_a of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of ⁷⁷Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.     

Organic solvent extraction of proteins and peptides from serum as an effective sample preparation for detection and identification of biomarkers by mass spectrometry

A method to extract peptides and low molecular weight proteins from serum under denaturing conditions using acetonitrile containing 0.1% trifluoroacetic acid has been developed. The extraction procedure precipitates large, abundant proteins to simplify subsequent mass spectral analysis. This sample preparation method provides an efficient way to extract serum peptides, enabling them to be compared and identified using different mass spectrometry approaches. Surface-enhanced laser desorption/ionization-time of flight mass spectrometry analysis of mouse blood serum samples prepared by this method allowed detection of two markers which were significantly reduced in mice with B cell lymphoma tumor. One of these markers has been identified as apolipoprotein A-II.