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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
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。
In some cases, proteins cannot be digested directly by enzymes
在某些情況下,
such as trypsin and require pretreatment prior
蛋白質無法直接被胰蛋白酶等酵素分解,
to digestion.
並在進行分解前需要預先處理。
One example of this is treatment of disulfide bonds,
其中一個例子是雙硫鍵的處理,
which are reduced and alkylated prior to digestion.
在分解之前被還原和烷基化。
If the amino acid sequence of the triptych peptide
如果三聯胜肽的氨基酸序列
is unique to the protein from which it was derived from,
對其來源的蛋白質是獨特的,
it is called the signature peptide.
則被稱為識別性胜肽。
The use of signature peptides means that the method
識別性胜肽表示其方法
is more selective and specific.
更為選擇性和特定性。
Triptych peptides should contain between 8 and 20 amino acids.
三聯胜肽應含有 8 至 20 個胺基酸。
In addition, a triptych peptide should not
此外,三聯胜肽不應含有
contain amino acids that can be easily chemically modified,
易於化學修飾的胺基酸,
such as cysteine and methionine.
像是半胱胺酸和甲硫胺酸。
The selection of triptych peptides
三聯胜肽的選擇將在
will be discussed in more detail in other modules
本系列的其他組建中
in this series.
進行更詳細的討論。
Now that we've covered the basic structure of peptides
現在我們已談到了胜肽和蛋白質的基本結構,
and proteins, and we've discussed
也已經討論過如何運用酵素分解
how peptides can be produced from proteins using
從蛋白質產出胜肽,
enzyme digestion, let's look at how peptides fragment
讓我們來看看
in a mass spectrometer.
胜肽是如何在質譜儀中碎斷的。
This slide highlights some of the differences
這張幻燈片凸顯了小分子的 LC-MS
between LC-MS of small molecules and LC-MS
與蛋白質及胜肽的 LC-MS 之間
of proteins and peptides.
的一些差異。
One difference, which has already
其中一項差異
been discussed in earlier slides,
已經在之前的幻燈片中討論過,
is that peptides form multiply charged ions.
就是胜肽會形成多電荷的離子。
Doubly, triply, and even high charge peptide ions
雙重、三重,甚至高電荷胜肽離子
are very common.
是非常常見的。
This is very different to small molecule LC-MS
這與先質離子通常帶單電荷的
where usually the precursor ion is singly charged.
小分子 LC-MS 非常不同。
Peptide fragments generated in a mass spectrometer collision
在質譜儀碰撞室中產生的胜肽片段
cell will have fewer charges then the precursor ions.
將具有較少的先質離子電荷。
This means that peptide fragments that
這表示具有較少電荷的胜肽片段
have fewer charges will appear at a higher mass
將以比先質離子更高的
to charge ratio than the precursor ions.
質荷比出現。
This is very different to what you
這與您在小分子片段中 所看到的非常不同,
would see in the small molecule fragmentation
在小分子片段中,
where the product ion is always at a lower mass to charge
產物離子的質荷比
ratio than the precursor ion.
總是會比先質離子低。
Also, as we've seen before, peptides fragment
另外正如我們之前所見,
in a highly predictable manner along the amino acid chain.
胜肽會以高度可預測的方式 在胺基酸鏈上碎斷。
Peptides can fragment at a number of predictable locations
胜肽可在胜肽鏈中的
in the peptide chain.
許多可預測位置上進行碎斷。
The nomenclature that result in fragment ions
導致片段離子的命名法
depend on which bond has been broken.
取決於哪個鍵被破壞。
When fragmentation occurs at the peptide bond,
當胜肽鍵發生碎斷時,
the C-terminal fragments is called
C 端片端稱為 y 離子,
the y ion and the N-terminal fragment is called the b ion.
N 端片段稱為 b 離子。
Y and b ions are the most important
Y 和 b 離子在使用質譜儀定量分析時
for quantification using mass spectrometry.
是最重要的。
For triptych peptides, the y ion will always
對於三聯胜肽,y 離子在 C 端
have a lysine or arginine amino acid at the C-terminal end.
總是有離胺酸或精胺酸胺基酸。
Fragmentation can also occur adjacent to the peptide bond,
碎斷也可能發生在胜肽鍵附近,
leading to other ions which are called z, c, a, and x ions.
造成稱為 z、c、a 和 x 離子的其他離子。
As we've already discussed, peptides
正如我們討論過的,
can produce a number of predictable fragment ions.
胜肽可以產生許多可預測的片段離子。
The selections that we're trying to use in an MRM experiment
我們在 MRM 實驗嘗試使用的選擇
need to be carefully considered.
必須仔細考慮。
In this example, fragmentation of the ion
在此例子中,
at 523.2808 results in a number of fragment ions
523.2808 的離子碎斷
shown in the lower half of the slide.
會導致在下半部幻燈片 顯示的許多片段離子。
Which ones would be the best to use in an MRM method?
MRM 方法中該用哪個最好?
There are a number of potential fragment ions we could use.
我們可以使用一些潛在的片段離子。
There's the most intense ion at 239--
239 有最強的離子 --
other ions at 341, 523, 873, 1045.
其他離子在 341、523、873、1045。
Let's evaluate these ions now.
現在我們來評估這些離子。
The ions shaded in red, although intense,
紅色的離子雖然強,
may not be a good choice as these are all low masses
但可能不是好的選擇,
and could be prone to interference
因為這些都是低質量的,
from other peptides.
可能容易受到其他胜肽的干擾。
The ion at 1045 is the singly charged ion from 5232,
1045 的離子是來自 5232 的單電荷離子,
so it would not be utilized.
因此不會被使用。
The y ion shown in the green shaded area at 873, 944, 802,
在 873、944、802 和 674 顯示的
and 674 are all potentially usable
綠色區域 y 離子全都可能可用,
as they are of sufficient intensity and size.
因為具有足夠的強度和大小。
This slide again highlights another feature
這張幻燈片再次凸顯了
of peptide fragmentation in a mass spectrometer, which
質譜儀中胜肽碎斷的另一個特徵,
is doubly charged ions fragmented
是雙電荷離子碎斷成單電荷離子,
to singly charged ions, therefore resulting
因此導致產物離子
in a product ion at a higher mass
的質荷比高於
to charge ratio than the precursor ion mass.
先質離子質量。
So we may not have access to standards
因此我們可能無法獲得
of all the potential triptych peptides we want
開發 MRM 方法想要的
to develop MRM methods for.
所有潛在三聯胜肽的標準。
However, there are software tools
但是有些如 Skyline 等
such as Skyline which can predict fragmentation
軟體工具可以預測
of triptych peptides.
三聯胜肽的碎斷。
Tools such as Skyline's prediction
Skyline 等工具的
suggest fragment ions that can be used in LC-MS method
預測建議片段離子
development.
可以使用於 LC-MS 方法開發。
These ions can be evaluated later by experiment.
這些離子可以透過實驗進行評估。
This is very important, as it means that you do not
這是非常重要的,
need to have access to standards of the triptych peptide
因為這表示您不需要取得三聯胜肽的標準
for initial method development.
來進行初始方法開發。
So let's summarize what we learned
因此讓我們總結一下
about peptide ionization or fragmentation.
我們對胜肽游離化或碎斷的了解。
Peptides form multiply charged ions,
胜肽會形成多電荷離子,
which is very different to traditional small molecule
這與傳統的小分子分析
analysis.
非常不同。
Peptides fragment in a highly predictable manner
質譜儀中的胜肽
in the mass spectrometer, and these fragments
以高度可預測的方式碎斷化,
can be predicted using software tools.
這些片段可使用軟體工具進行預測。
The software tools also recommend
此軟體工具也推薦
which MRM transition to use.
MRM 轉換使用。
The resultant fragment ions, which are often y ions,
產生的片段離子通常是 y 離子,
have a higher mass to charge than the precursor mass
會有比先質離子
to charge.
更高的質荷比。
The MRM transitions that are finally used
最終使用的 MRM 轉換
are selected based on specificity and intensity
是依據片段離子的特異性和強度
of the fragment ions.
進行選擇。
So let's summarize some of the key points of this introduction
那麼我們來總結一下這次
to peptides and protein structure.
介紹胜肽和蛋白質結構的重點。
Peptides and proteins are made of amino acids
胜肽和蛋白質是由胺基酸組成,
and can form a variety of complex structures.
可能形成各種複雜的結構。
Small proteins and peptides can be analyzed directly, i.e.,
小型蛋白質和胜肽可以直接進行分析,
intact by tandem quadrupole LC-MS systems.
即以串列四極 LC-MS 系統完整。
Larger proteins usually require digestion to smaller peptides
較大的蛋白質通常需要透過
for quantification by tandem quadrupole LC-MS systems.
串列四極 LC-MS 系統分解成 較小的胜肽來進行定量。
Enzymatic cleavage sites are predictable,
酵素切割位置是可以預測的,
and software tools are available that
有軟體工具可以
can predict triptych peptides.
預測三聯胜肽。
The structure of peptides and protein
胜肽和蛋白質結構會影響
impacts all stages of the bioanalysis workflow.
生物分析工作流程的各個階段。
This slide shows the workflow for the surrogate workflow
這張幻燈片顯示代表性 工作流程方法的流程,
approach, where a protein is enzymatically digested
其中蛋白質被胰蛋白酶酵素分解,
by trypsin to produce signature or unique peptides.
以產生識別性或獨特胜肽。
The process starts with selecting
此流程從選擇
unique peptides which represent the protein we
代表我們想測量的蛋白質的
are trying to measure.
獨特胜肽開始。
These unique peptides are predicted by software tools.
這些獨特胜肽由軟體工具進行預測。
The best MRM transitions are then selected and optimized.
接著選擇最佳的 MRM 轉換並優化。
We then go through the process of optimizing
然後我們會經過優化 一些準備工作的過程,
some for preparation, which may involve clean-up at the protein
可能涉及在蛋白質等級的清理、
level, reduction of colation, digestion, and peptide level
減少過濾、分解,
clean-up.
和胜肽等級的清理。
The MRM transitions, may then need to be fine
MRM 轉換可能需要使用生物基質中
tuned using peptides generated in a biological matrix.
產生的胜肽進行微調。
The structure of peptides and protein is an important factor
胜肽和蛋白質的結構是一個重要的因素,
and needs to be considered in all of the above steps.
需要在上述所有步驟中考慮。
This presentation was designed to introduce peptide
本次介紹旨在介紹胜肽和蛋白質的結構,
and protein structure and how the structure of peptide
以及胜肽和蛋白質的結構
and protein influences LC-MS method development.
如何影響 LC-MS 方法開發。
Further information is available on a variety of web based
更多資訊可在各種網路資源上取得,
resources, including these.
包括這些。
Thank you for listening.
感謝您的收聽。