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  • Hello and welcome to the next module

    哈囉,歡迎來到 Waters

  • in the Waters Peptide and Protein Bioanalysis Boot Camp.

    「胜肽和蛋白質生物分析 訓練營」的下一個單元

  • My name is Khalid Khan, and I'm part of Health Sciences'

    我是 Khalid Khan

  • marketing team here at Waters.

    Waters 健康科學行銷團隊的一員。

  • Today, I will be presenting on peptide and protein structure.

    今天我要介紹胜肽和蛋白質結構。

  • So let's get started.

    我們開始吧。

  • Here are a number of workflows for large molecule

    以下是大分子生物學療法

  • biotherapeutic and protein biomolecule analysis.

    和蛋白質生物分子分析的一系列工作流程

  • Today, LC-MS is increasingly used for protein quantification

    LC-MS 現在越來越常使用於蛋白質定量,

  • as an alternative to traditional ligand binding assays.

    作為傳統配體結合測定的替代方法。

  • Proteins can be analyzed by LC-MS,

    蛋白質可以透過 LC-MS 分析,

  • either using intact protein or surrogate peptide workflows.

    使用完整的蛋白質或代表性胜肽工作流程。

  • Both tandem quadrupole and high resolution mass spectrometers

    可以使用串列四極和

  • can be used.

    高解析度質譜儀。

  • Normal flow and microflow LC systems

    普通流量和微流量 LC 系統也

  • are also commonly used with both of these mass spectrometer

    常使用於這兩種

  • systems.

    質譜儀系統。

  • Most of this module will focus on the surrogate peptide

    此組件大部分專注於

  • workflow using tandem mass spectrometers,

    使用串列質譜儀的代表性胜肽工作流程,

  • and understanding your peptides and protein structure

    了解您的胜肽和蛋白質結構

  • is important when developing both intact and surrogate

    在開發完整和代表性胜肽工作流程

  • peptide workflows.

    都是非常重要的。

  • The areas covered in this presentation

    本次介紹所涵蓋的範圍為胺基酸、

  • will be the basic structure of amino acids,

    胜肽和蛋白質的基本結構,

  • peptides, and proteins, including

    包括一些具體的範例,

  • a few specific examples, such as monoclonal antibodies.

    如單株抗體等。

  • The basic structure of peptides and proteins

    胜肽和蛋白質的基本結構

  • has an impact on both the sample treatment and LC-MS method

    對樣本處理和 LC-MS 方法開發

  • development.

    都有影響。

  • The ionization and fragmentation of peptides

    胜肽的游離化和碎斷,

  • and how these aspects differ from small molecules

    以及在這些方面與小型分子的差別

  • will also be covered.

    都將提及。

  • The presentation is mainly intended

    本次介紹主要提供給

  • for scientists who already have some experience

    已經在小分子 LC-MS 方法開發上

  • in small molecule LC-MS method development.

    有些經驗的科學家。

  • Their aim is to provide an introduction to peptide

    他們的目標是提供

  • and protein structure and explain

    胜肽和蛋白質結構的介紹,

  • the commonly used terms in peptide protein LC-MS method

    並解釋在胜肽蛋白質 LC-MS 方法開發中

  • development.

    常用的術語。

  • The presentation will also prepare you

    本次介紹也將為您在

  • for subsequent modules in the Waters Peptide and Protein

    Waters 胜肽和蛋白質生物分析新兵訓練營

  • Bioanalysis Boot Camp.

    的後續組件做好準備。

  • In this first section, let's look at the structure

    在第一部分中,

  • of peptides and proteins.

    我們來看看胜肽和蛋白質的結構。

  • Peptides and proteins are chains of amino acids joined together.

    胜肽和蛋白質是連結在一起的胺基酸鏈。

  • There is no agreed criteria that specifies

    沒有規定胺基酸鏈長度的議定標準,

  • the length of an amino acid chain that defines whether it

    這會定義它被稱為

  • is called a peptide or protein.

    胜肽或蛋白質。

  • One common definition is that if the amino acid chain consists

    一種常見的定義是,

  • of less than 50 amino acids, it is

    如果胺基酸鏈由少於 50 個胺基酸組成,

  • called a peptide, and more than 50 amino acids,

    即稱為胜肽,

  • it is called a protein.

    超過 50 個胺基酸即稱為蛋白質。

  • This definition is not absolute, and you

    這項定義不是絕對的,

  • can have large peptides and small proteins

    您可以擁有相似胺基酸鏈長度的

  • of similar amino acid chain lengths.

    大型胜肽和小型蛋白質。

  • All of human proteins are formed from just 20

    所有的人類蛋白質

  • naturally occurring amino acids, or 21,

    都是由 20 種天然存在的氨基酸所組成的,

  • if you include selenocysteine.

    如果包含硒半胱氨酸的話是 21 種。

  • In terms of molecular weight, peptides

    就分子量而言,

  • are typically less than 6000 daltons,

    胜肽通常小於 6000 道耳頓,

  • whereas proteins can be anywhere from 5800

    而蛋白質則可能從像是胰島素等

  • daltons for a small protein such as insulin

    5800 道耳頓的小型蛋白質,

  • or several hundred thousand daltons for large proteins

    到甲狀腺球蛋白等

  • such thyroglobulin.

    幾十萬道耳頓的大型蛋白質都有。

  • This slide illustrates the mechanism

    此幻燈片說明了兩個胺基酸

  • of how two amino acids join together

    如何結合形成胜肽鍵

  • to form a peptide bond.

    的機制。

  • The carboxyl group of one amino acid

    胺基酸的羧基與

  • reacts with the amine group of another amino acid

    另一個胺基酸的胺基反應,

  • to form a peptide bond.

    形成胜肽鍵。

  • The resultant peptide will have a carboxyl group on one end,

    組成的胜肽在一端具有羧基,

  • and this is referred to as the C-terminal end.

    被稱為 C 端。

  • The amine group is referred to as the N-terminal end.

    胺基則被稱為 N 端。

  • As we will see later, these peptide bonds

    我們將在後面看到,

  • fragment in a highly predictable manner in a mass spectrometer

    這些胜肽鍵會在質譜儀碰撞室中

  • collision cell.

    以高度可預測的方式碎裂。

  • Amino acids and peptides can exist as zwitterions.

    胺基酸和胜肽可能以兩性離子形式存在。

  • This means that they can have both negative and positive

    這表示它們可能擁有負電和正電荷,

  • charges, depending on the pH.

    取決於 pH 值。

  • This is an important factor when developing sample clean up

    在胜肽層級開發樣本淨化方法時,

  • methods at the peptide level.

    這是一項重要的因素。

  • This will be discussed in more detail in later modules.

    這將在之後的組件中進行更詳細的討論。

  • The chain of amino acids that form the backbone of a peptide

    形成胜肽或蛋白質主鏈的氨基酸鏈

  • or protein is referred to as its primary structure.

    被稱為其主要結構。

  • Amino acids are usually represented by a single letter

    胺基酸通常以單一字母

  • or three letter abbreviation.

    或三個字母的縮寫表示。

  • Here is the table of the 21 amino acids

    這裡是形成人類胜肽和蛋白質

  • from which human peptides and proteins are formed.

    的 21 種胺基酸表格。

  • Some single letters are obvious, for example, G

    一些單一字母顯而易見的,

  • for glycine and A for alanine.

    比方說 G 代表甘胺酸(glycine), A 代表丙胺酸(alanine)。

  • Others are less obvious, such as K for lysine

    其他就沒那麼明顯了,

  • and R for arginine.

    像是 K 代表離胺酸,R 代表精胺酸。

  • As we will see later in this presentation,

    我們在這次介紹的後面會看到,

  • lysine and arginine are very important when

    在討論使用特定酵素將大型蛋白質

  • we discuss the breakdown of large proteins

    分解成較小的胜肽時,

  • into smaller peptides using specific enzyme digestion.

    離胺酸和精胺酸是非常重要的。

  • This slide illustrates the wide variety of structures

    這張幻燈片說明了

  • and resultant chemical properties of amino acids.

    各式各樣的氨基酸結構和化學性質。

  • The chemical structure of the amino acids

    胺基酸的化學結構

  • influences the polarity, hydrophobicity,

    會影響組成胜肽和蛋白質的

  • and acidic/basic nature of the resultant peptides

    極性、疏水性和

  • and proteins.

    酸 / 鹼性質。

  • Note that cysteine contains a sulfur atom, which

    請注意,光胱胺酸包含一個硫原子,

  • means that two cysteine amino acids can form

    這表示兩個光胱胺酸胺基酸之間

  • disulfide bonds between them.

    可以形成雙硫鍵。

  • These disulfide bonds can form in the same peptide chains

    這些雙硫鍵可以在相同的胜肽鏈中形成,

  • or connect two different peptide chains.

    或是連結兩個不同的胜肽鏈。

  • I stated earlier that the diverse properties of peptides

    之前我說過,胜肽和蛋白質的不同性質

  • and proteins have a large impact on the sample pretreatment

    對於樣品的預處理和 LC-MS 方法開發

  • and LC-MS method development.

    有很大的影響。

  • Note that some amino acids have a second amine group, which

    請注意,

  • means that they have multiple sites that

    某些胺基酸有第二個胺基,

  • can be protonated to form multiply charged,

    這表示它們有多個可以質子化的位置

  • positive ions.

    來形成多電荷的正離子。

  • As the structures of all amino acids are well known,

    由於所有胺基酸的結構都已被熟知,

  • it is possible to calculate the mass of a peptide

    因此有可能由其胺基酸成分

  • from its amino acid constituents.

    計算胜肽的質量。

  • Don't worry you will not have to calculate these manually.

    別擔心,您不必手動進行計算。

  • Software tools are available to do this automatically for you.

    有軟體工具可以自動幫您進行。

  • Software tools, such as Skyline, will automatically

    如 Skyline 等軟體工具

  • calculate the molecular weight of a peptide

    將自動從胺基酸序列

  • from its amino acid sequence.

    計算胜肽的分子量。

  • For example, the peptide D-E-V-I-L,

    舉例來說,由天冬胺酸、麩胺酸、

  • which consists of aspartic acid, glutamic acid, valine,

    纈胺酸、異白胺酸和白胺酸

  • isoleucine, and leucine, will have a mass of 587.31662

    組成的胜肽 D-E-V-I-L,

  • daltons.

    將具有 587.31662 道耳頓的質量。

  • Note that the table above lists the monoisotopic mass

    請注意,上面的表格列出了

  • and average mass.

    單一同位素質量和平均質量。

  • The monoisotopic mass is the mass where only the most

    單一同位素質量是在計算中

  • abundant isotopes are used in the calculation,

    只使用最豐富同位素的質量,

  • i.e., carbon-12, hydrogen-1, oxygen-16.

    即碳-12、氫-1、氧-16。

  • The average mass has all the minor isotopes

    平均質量將所有微量同位素

  • also included in the calculation, i.e., carbon-13,

    都納入計算,

  • deuterium, and nitrogen-15.

    即碳-13、氘和氮-15。

  • Proteins can exist in different forms and structures.

    蛋白質可以以不同的形式和結構存在。

  • So far, we have only discussed the basic amino acid

    目前我們只討論了

  • sequence, which is referred to as the primary structure.

    被稱為基本結構的鹼性胺基酸序列。

  • Amino acids can form hydrogen bond interactions

    胺基酸可在彼此之間

  • between each other, which influences the shape

    形成氫鍵交互作用,

  • of a peptide chain or protein.

    這會影響胜肽鏈或蛋白質的形狀。

  • The most common structures are a pleated sheet and half a helix.

    最常見的結構是褶板和半螺旋。

  • Bonds and interaction between alpha helices

    α 螺旋和褶板之間的鍵和交互作用

  • and pleated sheets result in tertiary structures.

    會導致三級結構。

  • Sulfa bonds between cysteine amino acids

    半胱胺酸胺基酸和胜肽鏈之間的

  • and the peptide chains are common in tertiary structures.

    磺胺鍵在三級結構中很常見。

  • Finally, when more than one different type of peptide chain

    最後,當涉及多種不同類型的胜肽鏈時

  • is involved, quaternary structure is produced.

    會產生四級結構。

  • This slide illustrates the primary structure

    此幻燈片說明了胰島素的主要結構,

  • of insulin, which includes two amino acid chains joined

    包含兩個結合在一起的胺基酸鏈,

  • together, the insulin A chain and the insulin B chain.

    胰島素 A 鏈和胰島素 B 鏈。

  • The diagram on this slide also shows

    此幻燈片上的圖表也顯示了

  • a diagram of the tertiary structure of insulin.

    胰島素的三級結構圖。

  • Here is an example of a peptide drug, desmopressin.

    這是胜肽藥物 desmopressin 的範例。

  • This is a relatively small peptide

    這是由九個胺基酸所組成的

  • comprised of nine amino acids.

    較小型胜肽。

  • LC-MS development of a peptide of this length

    這種長度的胜肽 LC-MS 開發

  • can be treated in the same way as a small molecule LC-MS

    可以用與小分子 LC-MS 相同的方式

  • method.

    進行處理。

  • The peptide can be analyzed directly

    該胜肽可以直接透過 LC-MS

  • by LC-MS and standards that are available for MRM method

    以及可用於 MRM 方法開發的標準

  • development.

    進行分析。

  • One difference from a small molecule ESI mass spectrum

    與小分子 ESI 質譜的一項不同

  • is the presence of a doubly charged positive ion

    在於除了單電荷離子之外

  • in addition to the singly charged ion.

    還存在雙電荷正離子。

  • This is a key feature of peptide ionization

    這是胜肽游離化的關鍵特徵,

  • that will be discussed later in this presentation

    之後將在本次介紹

  • and other modules.

    及其他組件中討論。

  • Note the doubly charged ion at 535.22

    請注意,535.22 的雙電荷離子

  • and the singly charged ion at 1069.435.

    和 1069.435 的單電荷離子。

  • An example of a small protein is insulin, which

    小型蛋白質的一個例子是胰島素,

  • consists of 51 amino acids.

    含有 51 個胺基酸。

  • The A chain has 21 amino acids, and the B chain

    A 鏈有 21 個胺基酸,

  • is 30 amino acids.

    B 鏈有 30 個胺基酸。

  • The monoisotopic mass of insulin is

    胰島素的單一同位素質量是 5023.6377,

  • 5023.6377, which is outside the range

    位於串列四極質譜儀系統的範圍之外,

  • of tandem quadrupole mass spectrometer systems which

    其通常擁有低於 2000 道耳頓的

  • typically have a maximum upper range of below 2000 daltons.

    最大上限範圍。

  • However, as insulin forms multiply charged ions

    但由於胰島素會形成

  • with three, four, and five charges,

    帶有三、四和五個電荷的多電荷離子,

  • it can be analyzed using tandem mass spectrometers.

    可以使用串列質譜儀進行分析。

  • In this example, the five plus ion is shown at mass 1162.

    在這個例子中, 五個正離子顯示為質量 1162。

  • Insulin also forms three plus and four plus ions.

    胰島素也會形成 3 及 4 離子。

  • Note again the disulfide bonds connecting the two amino acid

    請再注意在兩個現有胺基酸之間

  • chains between two existing amino acids.

    連結兩個胺基算鏈的雙硫鍵。

  • These are very common protein structures.

    這些是非常常見的蛋白質結構。

  • Here are some examples of larger proteins,

    這裡有些較大蛋白質的例子,

  • ranging from insulin like growth factor IGF-1

    從分子量為 7649 與胰島素類似的

  • with the molecular weight of 7649

    成長因子 IGF-1,

  • to thyroglobulin, which has a molecular weight over 660,000

    到分子量超過 660,000 道耳頓的

  • daltons.

    甲狀腺球蛋白。

  • The slide also shows medium sized proteins,

    此幻燈片也顯示中等大小的蛋白質,

  • such as CRP and apolipoprotein A1, which

    如 CRP 和脂蛋白元 A1,

  • have molecular weight in the mid 20,000 dalton range.

    其分子量在 20,000 道耳頓 的中等範圍內。

  • We can see that as the size of the proteins

    我們可以看到隨著蛋白質的大小增加,

  • increase, the challenge of measuring the intact protein

    測量完整蛋白質的挑戰就變得越加困難,

  • gets more difficult and is virtually

    使用範圍有限的串列質譜儀

  • impossible using limited range tandem mass spectrometers.

    更是實質上不可能。

  • However, we can break down large proteins into smaller peptide

    但我們可以將大型蛋白質

  • units and analyze these peptides using tandem mass

    分解為較小的胜肽單位,

  • spectrometers.

    並使用串列質譜儀分析這些胜肽。

  • This approach is called a surrogate peptide approach

    這種方法稱為代表性胜肽法,

  • and is widely used in protein bioanalysis and protein

    廣泛使用於蛋白質生物分析

  • biomarker research.

    和蛋白質生物標記研究。

  • Antibodies are a specific class of proteins

    抗體是具有共同結構的

  • with a common structure.

    特定類別蛋白質。

  • They are large Y-shaped proteins with two heavy chains and two

    它們是具有兩條重鏈和兩條輕鏈的

  • light chains.

    大型 Y 型蛋白質。

  • The heavy chains are linked to each other by disulfide bonds.

    重鏈透過雙硫鍵互相連結。

  • Sulfa bonds also link the light chains with the heavy chains.

    磺胺鍵也連結了輕鏈和重鏈。

  • Human immunoglobulins and antibody

    人類免疫球蛋白和抗體

  • produce white plasma cells to fight infections.

    會產生白血漿細胞來抵抗感染。

  • The heavy chains contains approximately 440 amino acids,

    重鏈含有約 440 個胺基酸,

  • and the light chains contain 220 amino acids.

    輕鏈含有 220 個胺基酸。

  • Monoclonal antibody drugs now form a very important class

    單株抗體藥物現在成為一種

  • of therapeutics and need to be measured in biomedical studies

    非常重要的療法,並需要在

  • and clinical research studies.

    生物醫學研究和臨床研究中進行量測。

  • One of the most widely used monoclonal antibody drugs

    目前使用最廣泛的單株抗體藥物

  • today is infliximab, which is used

    是 infliximab,

  • to treat autoimmune conditions such as Crohn's disease.

    用於治療克隆氏症等自身免疫病症。

  • Infliximab binds to TNF alpha and has

    Infliximab 與 TNFα 結合,

  • a molecular weight of approximately 150,000 daltons.

    擁有約 150,000 道耳頓的分子量。

  • Infliximab is known as a chimeric antibody.

    Infliximab 被稱為嵌合抗體。

  • Infliximab binds to TNF alpha.

    Infliximab 會與 TNFα 結合。

  • And infliximab is a chimeric antibody.

    infliximab 是一種嵌合抗體。

  • So how do we analyze large proteins

    那麼我們該如何使用

  • of several thousand daltons using tandem quadrupole mass

    通常質量限制範圍小於 2000 道耳頓的

  • spectrometers which usually have a limited mass range of less

    串列四極質譜儀來分析

  • than 2000 daltons.

    數千道耳頓的大型蛋白質呢。

  • The approach used is to break down the proteins

    運用的方法是使用酵素

  • into smaller peptides using digestion with enzymes.

    將蛋白質分解成較小的胜肽。

  • A number of different enzymes are used.

    使用多種不同的酵素。

  • The most commonly used enzyme is trypsin,

    最常用的酵素是胰蛋白酶,

  • which cleaves proteins in very specific locations.

    會在非常特定的位置切割蛋白質。

  • Trypsin cleaves proteins adjacent to lysine

    胰蛋白酶會在鄰近離胺酸和精胺酸的位置

  • and arginine.

    切割蛋白質。

  • Cleavage is always on the c-terminal side

    切割都會在

  • of the amino acid.

    胺基酸的 C 端進行。

  • This means that peptides arising from trypsin digestion, which

    這表示從胰蛋白酶分解產生的胜肽,

  • are called triptych peptides, can

    稱為三聯胜肽,

  • be predicted from the amino acid sequence of the protein.

    可從蛋白質的胺基酸序列被預測。

  • Online software tools are available to predict

    線上軟體工具可以預測

  • triptych peptides.

    三聯胜肽。

  • These online tools also predict the fragmentation

    這些線上工具也能預測