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

of those peptides in a mass spectrometer.

那些在質譜儀中的胜肽碎斷。

This is the basis of the surrogate peptide approach,

這是代表性胜肽法的基礎，

where a peptide or peptides are quantified

其中一種或多種胜肽

as a surrogate for the proteins from which

被定量為其派生自的

the peptides were derived from.

蛋白質代表者。