學(xué)霸歸納-蛋白質(zhì)與蛋白質(zhì)組

1、蛋白質(zhì)與蛋白質(zhì)組伍乘風(fēng)出品Chapter 1.Protein structure1. Regulation of protein function: Expression, Transportation and localization, Modification, Interaction, Degradation.2. Proteins generally have 50 amino acid residues.3. Levels of structure:1) Amino acid residue sequence2) Structural elements3) Specific 3-di

2、mensional structure4) Arrangement of subunits in multi-subunit protein4. There are over 300 naturally occurring amino acids on earth, but the number of different amino acids in proteins is only 20.5. Classification of amino acids:1) Acidic: aspartate(Asp, D), glutamate(Glu, E).2) Basic: lysine(Lys,

3、K), arginine(Arg, R), histidine(His, H).3) Aromatic: tyrosine(Tyr, Y), tryptophan(Trp, W), phenyl-alanine(Phe, F).4) Sulfur: cysteine(Cys, C), methionine(Met, M).5) Uncharged hydrophilic: serine(Ser, S), threonine(Thr, T), asparagine(Asn, N), glutamine(Glu, Q).6) Inactive hydrophobic: glycine(Gly, G

4、), valine(Val, V), leucine(Leu, L), isoleucine(Ile, I).7) Special structure: proline(Pro,P) is often located at the turn of a peptide chain.6. Interaction between positive and negative R groups may form a salt bridge, which is an important stabilizing force in proteins.7. The synthesis of all peptid

5、e chains starts from methionine.8. Two additional amino acids: selenocystein(硒半胱氨酸,UGA) and pyrrolysine(吡咯賴氨酸,UAG). The unusual amino acid is specified by a canonical(標(biāo)準(zhǔn)的) stop codon.9. The appropriate tRNA is initially charged with serine, which is subsequently modified to selenocysteine. However,

6、tRNA can be directly charged with pyrrolysine, which represents the first case of tRNA charging with a non-canonical amino acid.10. Trp, Tyr, and to a lesser exten Phe, absorb ultraviolet light, which accounts for the characteristic strong absorbance of light by proteins at a wave length of 280 nm.1

7、1. Nonstandard amino acid: Derived from one of the 20 standard amino acids, in a modification reaction that occurs after the standard amino acid has inserted into a protein.12. Three ways to obtain a peptide:1) Purification from tissue2) Genetic engineering3) Direct chemical synthesis13. Solid phase

8、 peptide synthesis(SPPS):1) Pre-treatment of amino acids2) Formation of peptide bond3) Deprotect, hydrolyze and purification14. Biological and chemical synthesis of peptides:l Common points1) Activation of carboxyl group2) Step by stepl Differences 1) Template2) Orientation: for biological synthesis

9、, it is from “N” to “C”. For chemical ways, it is from “C” to “N”.3) Efficiency4) Condition5) size15. Elucidation of antigenic peptide sequences in proteins by epitope mapping(表位作圖).16. Edman degradation1) Coupling: the N of the protein couples with PITC under basic conditions to form a PTC(phenylth

10、iocarbamyl苯氨基硫甲酰)-polypeptide.2) Cleavage: the peptide bond of the N PTC-residue undergoes acid cleavage from the polypeptide chain, releasing an unstable ATZ derivative of the amino acid.3) Conversion: the unstable ATZ-amino acid is converted into the corresponding stable PTH derivative.17. Large p

11、roteins sequencing:1) Breaking disulfide bonds2) Cleaving the polypeptide chain3) Sequencing of peptides4) Ordering peptide fragments5) Locating disulfide bonds18. A protein that is related to another by common evolutionary history(homologous proteins).19. The features of an helix:1) Every 3.6 resid

12、ues make one turn2) The distance between two turns is 0.54 nm3) The C=O of one turn is hydrogen bonded to N-H of the neighboring turn 4) An helix can be either right-handed or left-handed.20. -turns:1) a common structural element used to redirect the chain.2) A -turn is made of 4 residues. The carbo

13、nyl O fo the first residue is hydrogen bonded to the N of the fourth residue.21. Fibrous proteins:1) -keratin: coiled-coil of helices2) -keratin: stacked sheets. Alternating Ala-Gly allows close packing of Alas methyl group in silk.3) Collagen: usual sequence is repeats of Gly-X-(Hy)Pro. Three left-

14、handed helices wrap around each other in a right-handed spiral. The Glys are at core of bundles.22. Domains: the stable unit of protein folding and tertiary structure. Different domains in a protein may have different function.23. Advantages of multi-domains1) Efficient folding2) Larger folded prote

15、ins.3) Flexibility and motion4) Union of new function24. The force governing subunit association:1) Ionic interactions2) Hydrophobic interactions3) The secondary structure25. Reasons for using multimerc(多聚體的) assemblies of protomers(原體)：1) Allows larger protein without increasing genetic information

16、 required to code for it.2) Error rate is not such a factor3) Interaction sites can be at interface26. Try to predict structures based on the primary sequences, using:1) Known structures2) Tendencies of amino acids3) Energy minimization27. Measure/monitor protein folding1) CD(circular dichroism圓二色性)

17、 can measure conten of regular structures like helix and sheet.2) Trp fluorescence increases in hydrophobic environment.28. Forces acting on proteins1) Hydrogen bonding2) Van der Waals interactions3) Ion pairing4) Disulfide bonds5) Intrinsic properties6) Hydrophobicity: the dominant force in protein

18、 folding29. The relationship between primary structure and conformation:1) Change primary structurechange conformation2) Change primary structurewithout change of conformation3) Change conformationwithout change of primary structureChapter 2.Protein folding, misfolding and disease1. Protein folding

19、is the process by which a protein assumes its functional shape or conformation.2. Stringent(嚴(yán)格的) quality-control systems ensure that the misfolded products are targeted for degradation before the cause harm.3. Native states of proteins almost always correspond to the structures that are mos thermody

20、namically stable under physiological conditions.4. The folding process is a stochastic(隨機(jī)的) search of the many conformations accessible to a polypeptide. Finally find its lowest-energy structure by a process of trial and error.5. Larger proteins generally fold in modules(模塊). 6. Co-translational: fo

21、lding is initiated before the completion of protein synthesis.7. Post-translation: folding is initiated after release from the ribosome.8. Molecular chaperones are involved in folding to ensure that the various stages in the folding of such systems are all completed efficiently. They increase the ef

22、ficiency by reducing the probability of competing reactions, particularly aggregation.9. Peptidylprolyl isomerases increase the rate of cistrans isomerization of peptide bonds involving proline residues.10. Protein disulphide isomerases enhance the rate of formation and reorganization of disulphide

23、bonds.11. Function of chaperones(need ATP): 1. prevent misfolding; 2. Rescue misfolded and even aggregated proteins.12. The structural maturation of many proteins synthesized in the ER is slow and inefficient, probably because they require several post-translational modifications (for example, signa

24、l sequence cleavage, N-linked glycosylation, disulphide-bond formation and reshuffling, addition of glycosylphosphatidyl-inositol anchors, insertion of membrane proteins in the lipid bilayer).13. Quality-control in ER1) ER resident chaperones or oxidoreductases interact with intermediates who expose

25、 hydrophobic surfaces, unpaired cysteines or immature glycans, and as a consequence they are retained in the ER or retrieved from the Golgi complex.2) primary quality control monitors proteins architectural design through ubiquitous folding sensors.3) Secondary quality-control mechanisms rely instea

26、d on cell-specific factors and facilitate export of proteins.4) glycosylation and deglycosylation reactions enables correctly folded proteins to be distinguished from misfolded ones.14. Heat shock response and unfolded protein response induce the expression of genes encoding the protein chaperones a

27、nd folding catalysts. In this way, the cell up-regulates its protein-folding capacity.15. Protein turnover: Proteins within cells are continually being degraded to amino acids and replaced by newly synthesized proteins. This process is highly selective and precisely regulated.16. ubiquitinproteasome

28、 pathway: In eukaryotic cells, most proteins destined for degradation are labeled first by ubiquitin and then digested by the 26S proteasome.17. Many proteins of major medical importance (such as insulin) cannot fold properly in E. coli, are recognized as abnormal and are rapidly degraded. Successfu

29、l expression of such proteins required the development of mutant bacteria with reduced degradative capacity or improved expression systems that overwhelm the proteolytic systems.18. Bacteria have a specialized mechanism to target for destruction incomplete polypeptides while they are still on stalle

30、d ribosomes.19. The quality-control systems do not degrade all, or even most, mutated proteins: only those mutations that markedly perturb protein folding trigger rapid hydrolysis20. The degradation of free subunits of multimeric enzymes thus not only protects cells against accumulation of denatured

31、, potentially toxic polypeptides but also ensures that subunits of multimeric complexes are present in the proper proportion.21. ATP-dependent cytosolic systems exist in both bacteria and eukaryotic cells.It involves special protease in E. coli and ubiquitin-proteasome pathway in eukaryotic cells.22

32、. ER-associated degradation(ERAD): To maintain homeostasis, terminally misfolded molecules are retrotranslocated or dislocated(脫位的、移位的) across the ER membrane to be degraded by cytosolic proteasomes. 23. Common characteristics of amyloid deposits1) Specific optical behaviouron binding certain dye(染料

33、) molecules.2) The fibrillar structures with similar typical morphologies（形態(tài)） (long, unbranched and often twisted structures, a few nanometres in diameter).3) There are striking similarities in the aggregation behaviour of different peptides and proteins.Chapter 3. Regulation of protein functions1.

34、Expression: level; temporal; spatial2. Localization and translocation: most lysosomal, membrane, or secreted proteins have an amino terminal signal sequence.3. Common features of signal peptides:1) About 1015 hydrophobic amino acid residues2) 疏水殘基之前的N端含有1個(gè)或幾個(gè)帶正電的基團(tuán)。3) 靠近切割位點(diǎn)的C端含有小段極性短側(cè)鏈氨基酸。4. 運(yùn)輸?shù)骄€粒體

35、和葉綠體的蛋白的信號(hào)序列（signal sequence）被胞質(zhì)中的分子伴侶包裹，與靶細(xì)胞器外膜受體結(jié)合后通過(guò)蛋白通道進(jìn)入細(xì)胞器。其后，信號(hào)序列被切除。5. 核定位序列NLS可以位于肽鏈的任何位置，而且最終不會(huì)被切除。6. Modification: phosphorylation; glycosylation; methylation; acetylation; ubiquitination.7. 細(xì)胞通過(guò)對(duì)已存在的蛋白的翻譯后修飾來(lái)對(duì)外界環(huán)境作出反應(yīng)。8. Consequences of protein modifications: 1) Conformation change2) Tra

36、nslocation3) Protein interaction9. Interaction and dissociation10. DegradationChapter 4. Protein phosphorylation1. Phosphorylation refers to the addition of a phosphate to one of the amino acid side chains of a protein.2. Regulation by phosphorylation:1) Change the biological activity of an enzyme(由

37、于磷酸帶負(fù)電，與磷酸連接可以導(dǎo)致蛋白質(zhì)構(gòu)象發(fā)生變化)2) Helping move proteins between subcellular compartments3) Allowing interactions between proteins to occur4) Labeling proteins for degradation3. phosphorylation of the protein will act as a molecular switch, turning the activity on or off.4. The hydroxyl groups of Ser, Thr

38、, Tyr or His side chains are the most common target for protein kinases.5. 一些激酶與磷酸酶的特異性很強(qiáng)，另一些則能廣泛作用于很多蛋白。6. 利用磷酸化/去磷酸化來(lái)調(diào)節(jié)蛋白活性的優(yōu)勢(shì)：1) 迅速2) 不需要合成新蛋白或降解已有蛋白3) 簡(jiǎn)單可逆7. 外部信號(hào)可以激活蛋白激酶或磷酸酶，通過(guò)磷酸化級(jí)聯(lián)反應(yīng)引起一些列生理變化。動(dòng)物細(xì)胞中，這一級(jí)聯(lián)反應(yīng)由絲氨酸/蘇氨酸激酶和酪氨酸激酶調(diào)節(jié)。8. 磷酸化可能不改變蛋白的活性，而是提供磷酸化的位點(diǎn)與其他蛋白作用。例如在信號(hào)轉(zhuǎn)導(dǎo)過(guò)程中，一些膜蛋白磷酸化后與胞質(zhì)中的蛋白結(jié)合，從而起到了局

39、部富集的作用，是一些原本難以靠近的蛋白靠在一起，從而激活其他蛋白。9. 激酶的分類可以根據(jù)靶氨基酸的不同；底物的特異性和調(diào)節(jié)劑（regulators）的不同。10. 蛋白激酶的調(diào)節(jié)：1) 激活蛋白2) 抑制蛋白：假底物；自抑制（激酶的部分結(jié)構(gòu)域類似假底物）。3) 配體與調(diào)節(jié)亞基的結(jié)合4) 輔因子和第二信使5) 反式磷酸化（其他蛋白激酶）或順式/自磷酸化（自身）。6) 細(xì)胞內(nèi)的定位11. PKA：絲氨酸/蘇氨酸蛋白激酶；依賴cAMP；通過(guò)激活磷酸二酯酶進(jìn)行反饋性抑制?？烧{(diào)節(jié)糖和脂代謝。12. PKD：絲氨酸/蘇氨酸激酶，在生長(zhǎng)因子信號(hào)轉(zhuǎn)導(dǎo)途徑和壓力誘導(dǎo)信號(hào)途徑中扮演重要的角色。13. 受體酪氨酸

40、激酶：由跨膜受體和細(xì)胞內(nèi)側(cè)的激酶活性域組成。平時(shí)以單體形式存在，與配體結(jié)合后形成同源或異源二聚體，兩單體間相互磷酸化（反式-自磷酸化）發(fā)揮作用。14. Adaptor（銜接子） proteins are specialized protein binding partners that serve to link signaling proteins to each other, as a mechanism to propagate a cellular signal. Chapter 5. Glycosylation1. 糖基化的作用：1) 確保蛋白質(zhì)正確折疊2) 提高蛋白質(zhì)的穩(wěn)定性3)

41、參與細(xì)胞粘連（adhesion）4) 參與細(xì)胞信號(hào)傳遞2. 糖基化的共同特征：1) 需要酶催化2) 活化的核苷酸（如UDP）糖作為供體分子3) 具有位點(diǎn)特異性3. N-連接寡糖與Asn的側(cè)鏈中的氨基N相連。發(fā)生在Asn-Xaa-Ser/Thr這一特定序列上。其中Xaa可以是除Pro和Asp以外的所有氨基酸。4. N-聚糖可以分為“high mannose type”，“hybrid type”， “complex type”三類。都具有共同的5糖核心：Man（1，6）-（Man（1，3）-Man（1，4）-GlcNAc（1，4）-GlcNAc（1，N）-Asn。5. N-聚糖的三種類型都來(lái)自于

42、共同的前體（含有5糖核心），之后再通過(guò)修飾產(chǎn)生。6. N-聚糖的前體首先在長(zhǎng)萜醇上合成（在RER胞漿面開(kāi)始，后轉(zhuǎn)移至腔面），之后在轉(zhuǎn)移到肽鏈上（14糖前體）。（ER）。在高爾基復(fù)合體之中可被進(jìn)一步加工。7. N糖基化的程度取決于可用前體的數(shù)目、寡糖酰轉(zhuǎn)移酶的活性、Asn-X-Thr/Ser位點(diǎn)的數(shù)目以及其空間可用性。8. 粘蛋白O-連接聚糖（mucin-type O-linked glycans）一般出現(xiàn)在分泌或膜結(jié)合蛋白上。9. 粘蛋白O糖基化發(fā)生在高爾基體中，將N-乙酰半乳糖胺（連有其他糖）連接到絲氨酸或蘇氨酸上。10. 粘蛋白O糖基化的特點(diǎn)：1) 沒(méi)有特定的氨基酸序列。2) 具有組織特異

43、性。3) 由于發(fā)生在蛋白質(zhì)翻譯和折疊的后期，所以只有暴露的絲氨酸/蘇氨酸被糖基化4) 在粘蛋白O糖基化中，蘇氨酸比絲氨酸糖基化更加高效。11. O糖基化的作用：1) 維持糖蛋白的結(jié)構(gòu)2) 參與細(xì)胞識(shí)別（ABO血型）3) 調(diào)節(jié)酶和信號(hào)分子的活性4) 參與蛋白表達(dá)和加工5) 參與細(xì)胞內(nèi)的相互作用12. O-GlcNAc是廣泛存在于核質(zhì)蛋白上的單糖糖基化。糖基化可逆。N-乙酰-葡萄糖胺酶催化糖基的移除。不識(shí)別特定的序列，不過(guò)多發(fā)生在被激酶識(shí)別的位點(diǎn)。不修飾酪氨酸。13. O-GlcNAc可能和磷酸化一起起到調(diào)節(jié)作用，參與信號(hào)轉(zhuǎn)導(dǎo)。涉及轉(zhuǎn)錄、翻譯、腫瘤、神經(jīng)病理等生物過(guò)程。Chapter 6. Ubi

44、quitinylation and protein degradation1. 蛋白降解的意義：1) 為新蛋白合成提供氨基酸2) 除去過(guò)量的酶3) 除去不需要的轉(zhuǎn)錄因子2. 溶酶體處理細(xì)胞外蛋白和細(xì)胞表面膜蛋白（參與受體介導(dǎo)的內(nèi)吞作用）。3. 蛋白酶體處理內(nèi)生的蛋白質(zhì)。如轉(zhuǎn)錄因子，周期蛋白，病毒或其他胞內(nèi)寄生蟲(chóng)的蛋白，錯(cuò)誤折疊的蛋白等。4. 蛋白酶體為空心圓柱狀，活性中心位于柱內(nèi)部。包含核心顆粒（20S）和調(diào)節(jié)顆粒（19S）兩部分。核心顆粒具有蛋白酶活性，構(gòu)成柱身，調(diào)節(jié)顆粒位于柱兩端，識(shí)別泛素標(biāo)記的蛋白，破壞蛋白的三級(jí)結(jié)構(gòu)，將其誘導(dǎo)如柱內(nèi)部核心顆粒除。5. 免疫蛋白酶體具有11S調(diào)節(jié)顆粒，具有

45、加工I類MHC肽的能力。6. 泛素是含有76個(gè)氨基酸的小蛋白質(zhì)，其序列在整個(gè)生物王國(guó)中高度保守。被所有生物用來(lái)標(biāo)記需要降解的蛋白質(zhì)。首先合成一個(gè)大的polyprotein，之后在切割為一個(gè)個(gè)小的泛素。7. 泛素可以一單體存在，也可以與其他泛素形成鏈狀結(jié)構(gòu)。其C端甘氨酸與靶蛋白的賴氨酸側(cè)鏈形成異肽鍵。被泛素鏈標(biāo)記的蛋白將會(huì)被26S蛋白酶體降解。8. 泛素化的過(guò)程：a) 泛素的活化：1) E1酶（泛素活化酶）催化ATP水解導(dǎo)致泛素腺苷化，形成Ub-AMP，同時(shí)放出PPi。2) 之后，另一個(gè)泛素的C端與E1活性位點(diǎn)處的半胱氨酸形成硫酯鍵。即E1-S-co-Ub.b) 泛素接合ubiquitin co

46、njugation：E1酶以硫酯鍵結(jié)合的泛素被轉(zhuǎn)移到E2酶（泛素接合酶）的半胱氨酸上，形成E2-s-co-Ub。c) 最后，由泛素-蛋白連接酶E3催化E2上的活化泛素轉(zhuǎn)移到蛋白質(zhì)上。9. 一些蛋白質(zhì)具有被稱為破壞盒（destruction box）的特殊序列，被相應(yīng)的泛素連接酶識(shí)別。10. 多聚泛素一般通過(guò)Lys48與Gly76形成異肽鍵產(chǎn)生。Lys48被半胱氨酸取代導(dǎo)致多聚泛素?zé)o法形成將導(dǎo)致蛋白質(zhì)無(wú)法被標(biāo)記降解。多聚泛素可以在蛋白質(zhì)上連接形成，也可以先合成好后再連接蛋白質(zhì)。11. 一般認(rèn)為，4個(gè)泛素連接形成的鏈能標(biāo)記蛋白質(zhì)被26S蛋白酶體水解。12. 除了Lys48外， Lys11和Lys2

47、9形成的多聚泛素也可以標(biāo)記蛋白質(zhì)被蛋白酶體水解。而Lys6和Lys63形成的多聚泛素不標(biāo)記蛋白質(zhì)降解，但可以改變蛋白質(zhì)活性和定位。13. 一條多聚泛素鏈與蛋白質(zhì)相連足以標(biāo)記其被降解。多條多聚泛素鏈與一個(gè)蛋白的連接的作用尚不清楚。第一章 緒論1. 傳統(tǒng)蛋白質(zhì)研究技術(shù)：測(cè)序，需要純化蛋白質(zhì)，一個(gè)純化樣品只能確定一個(gè)蛋白。2. 高通量技術(shù)：2D電泳+MS,不需要純化蛋白質(zhì)，可以從一個(gè)為純化樣品中確認(rèn)上千個(gè)蛋白。3. 蛋白質(zhì)組學(xué)研究是整體， 比較蛋白質(zhì)的動(dòng)態(tài)變化，研究其相互作用和功能。采用的技術(shù)高通量、自動(dòng)化。而傳統(tǒng)蛋白質(zhì)研究研究的是蛋白質(zhì)個(gè)體，分析其靜態(tài)性質(zhì)和結(jié)構(gòu)，采取小規(guī)模、非連續(xù)的技術(shù)。4. 蛋

48、白質(zhì)組學(xué)研究的重要性：1) 蛋白質(zhì)執(zhí)行幾乎所用生物功能的控制2) 蛋白間的相互作用控制絕大多數(shù)細(xì)胞發(fā)育過(guò)程3) 疾病在蛋白質(zhì)水平上發(fā)生并被處理4) 蛋白質(zhì)是藥物的靶點(diǎn)或被制成藥物。5. 蛋白質(zhì)組學(xué)的研究角度：1) 從蛋白質(zhì)化學(xué)角度對(duì)蛋白質(zhì)進(jìn)行研究2) 從生物學(xué)角度對(duì)差異蛋白質(zhì)進(jìn)行研究6. 蛋白質(zhì)組學(xué)研究的內(nèi)容：1) 特定細(xì)胞、組織、器官制造的蛋白質(zhì)種類2) 明確蛋白質(zhì)關(guān)系網(wǎng)絡(luò)3) 描繪蛋白質(zhì)三維結(jié)構(gòu)，確定其結(jié)構(gòu)上的關(guān)鍵部位。7. 蛋白質(zhì)分離技術(shù)：1) 雙向凝膠電泳；2) 二維/多維分離技術(shù)：親和色譜、反相色譜、毛細(xì)管電泳技術(shù)；分離度高、耗樣量少、易于與MS連接。3) 蛋白芯片技術(shù)。8. 蛋白質(zhì)

49、分析鑒定技術(shù)：生物質(zhì)譜，基質(zhì)輔助激光解吸電離MALDI和電噴霧電離ESI。第二章 蛋白質(zhì)樣品制備1. 樣品制備原則：1) 使蛋白樣品全部處于溶解狀態(tài)2) 防止樣品在聚焦時(shí)出現(xiàn)聚集和沉淀3) 防止制備過(guò)程中發(fā)生樣品的抽提后化學(xué)修飾（降解等）。4) 完全除去樣品中的核酸，盡量除去干擾蛋白5) 制備方法具有可重現(xiàn)性2. 樣品制備步驟：1) 分離：將生物提取液中蛋白與其他物質(zhì)分開(kāi)2) 提取：從總的蛋白質(zhì)溶液中提取目標(biāo)蛋白3) 純化：分離出雜質(zhì)使蛋白質(zhì)單一化3. 蛋白質(zhì)分離具體過(guò)程：1) Cell washing：需要緩沖液，防止細(xì)胞裂解2) Cell disruption3) Removal of c

50、ontaminant4) Microdialysis（微量透析）5) Electrophoretic（電泳的） desalting6) Precipitation（沉淀） methods：原理是加入適當(dāng)?shù)脑噭蛩徜@、三氯乙酸、丙酮）使蛋白質(zhì)分子處于等電點(diǎn)或失去水化層，從而使蛋白交替不再穩(wěn)定，而產(chǎn)生沉淀?？梢愿患蜐舛鹊鞍祝ルs質(zhì)同時(shí)抑制蛋白酶活性。7) Protease inhibitor：可利用變性劑、低溫、高pH等。8) DNA、RNA去除：核酸可被銀染，它們?cè)斐闪嗽谀z中出現(xiàn)水平條紋，它們使蛋白質(zhì)沉淀，去除：蛋白質(zhì)沉淀、核酸酶處理、超聲波處理、核酸提?。ㄈ绫椒樱?) 脂質(zhì)、多糖、固體雜

51、質(zhì)、鹽離子等，洗滌劑、沉淀、離心等10) 微量透析、電洗脫11) 一些不適合，電泳前在低電壓下（100V）5小時(shí)12) 蛋白質(zhì)沉淀：聚集蛋白、同時(shí)去除多種干擾雜質(zhì)、蛋白酶活動(dòng)抑制13) 離心獲得蛋白質(zhì)，但沉淀劑會(huì)影響凝膠電泳，會(huì)產(chǎn)生修飾14) 原理：加入適當(dāng)?shù)脑噭┦沟鞍踪|(zhì)分子處于等電點(diǎn)狀態(tài)或失去水化層，蛋白質(zhì)的膠體溶液就不在穩(wěn)定并將產(chǎn)生沉淀。15) 五種方法：硫酸銨、三氯乙酸、丙酮、三氯乙酸/丙酮、醋酸銨16) 抑制蛋白質(zhì)降解方法17) 強(qiáng)變性劑 (8 molL urea，10TCA或2 SDS) 18) 低溫:蛋白酶 低溫下無(wú)活性 1) 高pH:蛋白酶 pH超過(guò)9.0的時(shí)候失活。因此，在樣

52、品裂解液中出現(xiàn)Tris、碳酸鈉和載體兩性電解質(zhì)時(shí)也可以抑制蛋白降解4. 從天然物質(zhì)中提取蛋白質(zhì)的關(guān)鍵步驟包括：細(xì)胞裂解、pH控制、溫度控制、避免蛋白降解。5. 樣品的溶解是2-DE成功分離蛋白質(zhì)的最關(guān)鍵因素之一，可通過(guò)使用變性劑、表面活性劑和還原劑、起載體作用的兩性電解質(zhì)等增加樣品溶解性。6. 溶解的目標(biāo)：1) 破壞樣品中非共價(jià)結(jié)合的蛋白質(zhì)復(fù)合物和聚積體，從而形成各個(gè)多肽的溶解液。2) 必須允許可能干擾2-DE分離的鹽、脂類、多糖和核酸等物質(zhì)的去除。3) 保證樣品在電泳過(guò)程中保持溶解狀態(tài)。7. 亞細(xì)胞器的分離可通過(guò)差速離心及密度梯度離心。8. 順序抽提法：根據(jù)細(xì)胞蛋白溶解性的差異，用具有不同溶

53、解能力的蛋白溶解液進(jìn)行抽提的方法。9. 用離心技術(shù)分離出細(xì)胞器、質(zhì)膜和細(xì)胞核等成分，再用適當(dāng)?shù)牡鞍踪|(zhì)溶解液進(jìn)行分離?？梢詼p少樣品的復(fù)雜性，且有利于蛋白質(zhì)的定位。但需要專業(yè)的儀器，可能出現(xiàn)假陽(yáng)性。10. 細(xì)胞分離可以采用激光捕獲微解剖的方法。11. 對(duì)于高疏水性的蛋白，可以使用硫脲、SDS（緊急情況下，上限2%，需至少稀釋SDS樣品20倍與尿素折疊，用它的原因：阻止形成低聚物，溶解細(xì)胞間質(zhì)，溶解疏水蛋白），或使用新型的兩性離子洗滌劑。12. 低豐度蛋白的分離：除增加上樣量和提高總蛋白溶解度外。可采用預(yù)分級(jí)+窄pH膠的微制備技術(shù)進(jìn)行分離。13. 殘留的裂解液、鹽離子以及一些內(nèi)源性的小分子會(huì)對(duì)蛋白質(zhì)

54、的分離或電泳產(chǎn)生很大影響，應(yīng)當(dāng)盡可能除去。14. 蛋白質(zhì)含量測(cè)定：1) 雙縮脲試劑Biuret method（肽鍵）（紫色雙縮脲復(fù)合物）（大于1-20mg）2) Lowrys法(Folin-酚試劑法)（氮）與銅在堿性條件下反應(yīng)，大于0.1mg3) Bradford methods.（考馬斯亮藍(lán)）最大吸收峰465-595，大于10-100微克4) 紫外線技術(shù)（芳香基團(tuán)，280nm和200nm光吸收）280nm，大于0.1-1mg5) 特殊技術(shù)（蛋白質(zhì)中的特殊基團(tuán)，如血紅素等）6) 商業(yè)化的技術(shù)：BCA、DC、DC/RC。7) 出現(xiàn)問(wèn)題：裂解液影響聚焦（去污劑重泡脹溶液稀釋含SDS樣品，丙酮沉淀去

55、除SDS，苯酚需要去除）、鹽濃度影響（透析、旋轉(zhuǎn)透析、凝膠過(guò)濾、沉淀/重懸法脫鹽）、內(nèi)源性小分子影響（核酸，用酶處理，多糖，用硫酸銨、苯酚/醋酸銨沉淀，離心，脂肪，用變性劑和去污劑，不溶物質(zhì)，去除）第三章 雙向電泳技術(shù)1. 電泳技術(shù)圍繞制膠、電泳、染色三個(gè)技術(shù)環(huán)節(jié)進(jìn)行改進(jìn)，以實(shí)現(xiàn)以下目標(biāo)：1) 提高分辨率及靈敏度2) 簡(jiǎn)化操作，縮短電泳時(shí)間3) 擴(kuò)大應(yīng)用范圍2. V：泳動(dòng)速度；d：泳動(dòng)距離；t：電泳時(shí)間；E：電場(chǎng)強(qiáng)度；u：泳動(dòng)率/泳動(dòng)度（cm/vs/min）；U：電壓；l：電場(chǎng)的長(zhǎng)度。3. 電泳類型：1) 區(qū)帶電泳：支持物為固體，如紙電泳、醋酸纖維素薄膜電泳、粉末電泳、細(xì)絲電泳、凝膠電泳。2)

56、 自由界面電泳：支持物為溶液，很少用。4. 毛細(xì)管電泳是一類以毛細(xì)管為分離通道、以高壓直流電場(chǎng)為驅(qū)動(dòng)力的新型液相分離技術(shù)。毛細(xì)管電泳實(shí)際上包含電泳、色譜及其交叉內(nèi)容，它使分析化學(xué)得以從微升水平進(jìn)入納升水平，并使單細(xì)胞分析，乃至單分子分析成為可能。5. 聚丙烯酰胺凝膠電泳PAGE：?jiǎn)误w為丙烯酰胺Acr，交聯(lián)劑為N,N-甲叉雙丙烯酰胺Bis，加速劑為N,N,N,N-四甲基乙二胺TEMED，催化劑為過(guò)硫酸銨AP或核黃素。6. 凝膠電泳的物理效應(yīng)：樣品濃縮效應(yīng)（凝膠系統(tǒng)的不連續(xù)性產(chǎn)生）；分子篩效應(yīng)；電荷效應(yīng)。7. 蛋白質(zhì)的等電點(diǎn)（pI）：當(dāng)某蛋白質(zhì)在一定的pH的溶液中，所帶的正負(fù)電荷相等，它在電場(chǎng)中既

57、不向陽(yáng)極也不向陰極移動(dòng)，此時(shí)溶液的pH值叫做該蛋白質(zhì)的等電點(diǎn)。8. 取決于其氨基酸的組成，是一個(gè)物理化學(xué)常數(shù)。 9. 蛋白質(zhì)的帶電性質(zhì)與溶液的pH有關(guān)10. 不同Pr其aa的數(shù)量、種類及占比例不同，pI不同11. 基本原理：利用蛋白質(zhì)分子或其他兩性分子等電點(diǎn)的不同，在一個(gè)穩(wěn)定的、連續(xù)的、線性的（或非線性）pH梯度中進(jìn)行蛋白質(zhì)的分離和分析。12.13. 等電聚焦電泳IEF/EF：在一個(gè)穩(wěn)定的、連續(xù)的、（非）線性的pH梯度中根據(jù)蛋白質(zhì)的等電點(diǎn)進(jìn)行蛋白質(zhì)的分離和分析。14. 等電聚焦的分辨率用兩個(gè)鄰近帶的pI差來(lái)表示。其中D是蛋白的擴(kuò)散系數(shù)。分號(hào)上方中括號(hào)內(nèi)表示pH梯度；下方中括號(hào)內(nèi)表示蛋白質(zhì)遷移率的斜率。du/d(pH)是常數(shù)。所以只有通過(guò)提高場(chǎng)強(qiáng)和使用窄pH范圍來(lái)提高分辨率。但電壓過(guò)高會(huì)導(dǎo)致產(chǎn)熱過(guò)多使蛋白質(zhì)變性甚至燒膠。15. pH梯度的建立：(等電聚焦技術(shù)的關(guān)鍵)1) 載體兩性電解質(zhì)pH梯度CA：在電場(chǎng)中通過(guò)兩性緩沖離子建立的線性pH梯度。2) 固相pH梯度IPG：將緩沖基團(tuán)共價(jià)結(jié)合在介質(zhì)上成為