Tandem mass spectrometry (MS-MS) provides a rapid method for determining the amino acid sequence of peptides. Peptides undergo fragmentation reactions in the mass spectrometer, often accompanied by the breaking of amide bonds, forming so-called "b ions" and "y ions". The following shows the b and y ions formed in the tandem mass spectrum of alanine-valine-glycine (Ala-Val-Gly).

<MultiModal>![Alanine-valine-glycine (NC(C(NC(C(C)C)C(NCC(O)=O)=O)=O)C), which can be fragmented in secondary mass spectrometry to produce four b and y ions, where b1 ion is NC(C)C#[O+], y2 ion is [NH3+]C(C(C)C)C(NCC(O)=O)=O, b2 ion is NC(C(NC(C(C)C)C#[O+])=O)C, y1 ion is [NH3+]CC(O)=O](/media/uploads/ca6ff1fd-a920-4cc9-a22e-272014ece745.png)</MultiModal>

It is known that peptide **X** is an octapeptide, in which one amino acid has been modified, and this modification will not be reflected in the amino acid composition determination after hydrolysis. Tandem mass spectrometry was used to sequence peptide **X**. The mass-to-charge ratios of the formed b and y ions are given in the table below:

| Ion | m/z   | Ion | m/z   | Ion | m/z   |
| :--- | :---- | :--- | :---- | :--- | :---- |
| y1   | 90.1  | b3   | 298.1 | b6   | 598.2 |
| b1   | 112   | y4   | 374.2 | y6   | 618.3 |
| b2   | 169.1 | y7   | 675.3 | b7   | 697.3 |
| y2   | 189.1 | b4   | 413.1 | y5   | 489.2 |
| y3   | 246.1 | b5   | 541.2 | b8   | 768.3 |

The following statements are now available:

1.  The composition of the polypeptide is: 1 Ala, 1 Asp, 1 Gly, 2 Glu, 1 Val and 1 modified amino acid

2.  There are a total of 20160 kinds of polypeptides (including **X** itself) with the same amino acid composition as polypeptide **X** but different amino acid sequences.

3.  The modified amino acid is produced by Asp or Asn

4.  The modified amino acid is located at the C-terminus

5.  The polypeptide has 2 free carboxyl groups

6.  The molecular weight of the modified amino acid is 129

7.  The polypeptide can be sequenced by Edman degradation

8.  The polypeptide sequence is Mod-Gly-Glu-Asp-Gln-Gly-Val-Ala, where Mod refers to the modified amino acid

Select all the correct statements.
串联质谱技术（MS-MS）为测定多肽氨基酸次序提供了快捷的方法。多肽在质谱仪中会发生碎片化反应，碎裂时经常伴随着酰胺键的断裂，形成所谓的“b离子”和“y离子”。下面给出丙氨酰-缬氨酰-甘氨酸（Ala-Val-Gly）分子在串联质谱中形成的b离子和y离子。

<MultiModal>![丙氨酰-缬氨酰-甘氨酸（NC(C(NC(C(C)C)C(NCC(O)=O)=O)=O)C），可以在二级质谱中碎裂产生四种b、y离子，其中b1离子为NC(C)C#[O+]，y2离子为[NH3+]C(C(C)C)C(NCC(O)=O)=O，b2离子为NC(C(NC(C(C)C)C#[O+])=O)C，y1离子为[NH3+]CC(O)=O](/media/uploads/ca6ff1fd-a920-4cc9-a22e-272014ece745.png)</MultiModal>

已知多肽**X**是一个八肽，其中的一个氨基酸被修饰过，这种修饰在水解后的氨基酸组成测定中不会体现出来。使用串联质谱对多肽**X**测序。形成的b离子和y离子的质荷比在下表中给出：

| 离子 | m/z   | 离子 | m/z   | 离子 | m/z   |
| :--- | :---- | :--- | :---- | :--- | :---- |
| y1   | 90.1  | b3   | 298.1 | b6   | 598.2 |
| b1   | 112   | y4   | 374.2 | y6   | 618.3 |
| b2   | 169.1 | y7   | 675.3 | b7   | 697.3 |
| y2   | 189.1 | b4   | 413.1 | y5   | 489.2 |
| y3   | 246.1 | b5   | 541.2 | b8   | 768.3 |

现有以下说法：

1.该多肽的组成为：1个Ala，1个Asp，1个Gly，2个Glu，1个Val和1个被修饰的氨基酸

2.与多肽**X**氨基酸组成相同但氨基酸次序不同，共可组成20160种多肽（包含**X**）本身

3.被修饰的氨基酸是由Asp或Asn产生的

4.被修饰的氨基酸位于C端

5.该多肽有2个游离羧基

6.被修饰氨基酸的分子量为129

7.该多肽可以通过Edman降解法进行测序

8.该多肽序列为Mod-Gly-Glu-Asp-Gln-Gly-Val-Ala，其中Mod指代被修饰的氨基酸

选出其中所有正确的说法。
1, 2, 3, 4, 7
1, 2, 4, 6, 7
2，5，6，8
2, 5, 6, 7, 8
1, 2, 3, 4, 5
1, 2, 3, 4, 5, 7
3, 5, 8
2, 3, 5, 8
3, 4, 7
3, 4, 5, 7
2, 3, 4, 5, 7
2, 3, 5, 6, 8
All of the above options are incorrect.
1，2，3，4，7
1，2，4，6，7
2，5，6，8
2，5，6，7，8
1，2，3，4，5
1，2，3，4，5，7
3，5，8
2，3，5，8
3，4，7
3，4，5，7
2，3，4，5，7
2，3，5，6，8
以上选项均不正确
According to the given picture information, it can be categorized that: In tandem mass spectrometry, the fragmentation of peptide bonds in a polypeptide, where fragments containing the acyl portion produce acyl positive ions, are called b ions; fragments containing the amino portion produce amino positive ions, are called y ions.

<Checkpoint>[In tandem mass spectrometry, the fragmentation of peptide bonds in a polypeptide, where fragments containing the acyl portion produce acyl positive ions, are called b ions; fragments containing the amino portion produce amino positive ions, are called y ions.](1)</Checkpoint>

Therefore, the mass-to-charge ratio of the b ion plus 17 is the molecular weight of the corresponding amino acid, and the mass-to-charge ratio of the y ion minus one is the molecular weight of the corresponding amino acid. At the same time, increasing the sequence number of the b ion is equivalent to adding amino acids from the N-terminus of the polypeptide; increasing the sequence number of the y ion is equivalent to adding amino acids from the C-terminus of the polypeptide.

<Checkpoint>[The mass-to-charge ratio of the b ion plus 17 is the molecular weight of the corresponding amino acid, and the mass-to-charge ratio of the y ion minus one is the molecular weight of the corresponding amino acid](1)</Checkpoint>

<Checkpoint>[Increasing the sequence number of the b ion is equivalent to adding amino acids from the N-terminus of the polypeptide; increasing the sequence number of the y ion is equivalent to adding amino acids from the C-terminus of the polypeptide](1)</Checkpoint>

Analyzing the table data, the molecular weight of the amino acid corresponding to the y1 ion is 89, which is Ala; the molecular weight of the amino acid corresponding to the b1 ion is 129, which does not correspond to any amino acid, and should be a modified amino acid, statement 6 is correct.

<Checkpoint>[The molecular weight of the amino acid corresponding to the b1 ion is 129, which should be a modified amino acid](1)</Checkpoint>

The molecular weight of the polypeptide corresponding to the y2 ion is 188.1, subtracting the molecular weight of the Ala residue is 117.1, which is Val; the molecular weight of the polypeptide corresponding to the b2 ion is 186.1, subtracting the molecular weight of the modified amino acid residue is 75.1, which is Gly; the molecular weight of the polypeptide corresponding to the y3 ion is 245.1, subtracting the molecular weight of the polypeptide corresponding to the y2 ion is 57, corresponding to the mass of the amino acid residue, and adding a molecule of water is 75, corresponding to the mass of the amino acid, which is Gly. Subsequent b and y ions are analyzed using the same method, and the polypeptide sequence can be obtained as Mod-Gly-Glu-Asp-Gln-Gly-Val-Ala, where Mod refers to the modified amino acid, and the modified amino acid is located at the N-terminus. Therefore, statement 8 is correct and 1 is incorrect.

<Checkpoint>[The polypeptide sequence is Mod-Gly-Glu-Asp-Gln-Gly-Val-Ala, the modified amino acid is located at the N-terminus](2)</Checkpoint>

For the composition of polypeptide **X**, there are a total of $8!/2! = 20160$ possible sequences, statement 2 is correct.

<Checkpoint>[For the composition of polypeptide **X**, there are a total of $8!/2! = 20160$ possible sequences](1)</Checkpoint>

The modified amino acid is the same as the original amino acid after hydrolysis, which is a hydrolyzable modification, considered as acylation modification;

<Checkpoint>[Consider the amino acid modification as acylation modification](1)</Checkpoint>

Comparing Ser, Tyr, Thr, Arg, Lys, Cys, Asn/Asp, Gln/Glu, which can produce acylation modifications, the difference value is only 17/18 when it is Gln/Glu, corresponding to a molecule of ammonia or water, then the modified amino acid is pyroglutamic acid, statement 3 is incorrect.

<Checkpoint>[The modified amino acid is produced by Gln or Glu, modified as pyroglutamic acid](1)</Checkpoint>

The pyroglutamic acid amino group and the side chain carboxyl group are acylated to form a ring, and the N-terminus is blocked, so it cannot be sequenced by Edman degradation, statement 7 is incorrect.

<Checkpoint>[The pyroglutamic acid amino group and the side chain carboxyl group are acylated to form a ring, and the N-terminus is blocked, so it cannot be sequenced by Edman degradation.](1)</Checkpoint>

There are a total of three free carboxyl groups in polypeptide **X**: the C-terminal carboxyl group, one Asp side chain carboxyl group, and one Glu side chain carboxyl group, but the free carboxyl group of Asp forms a ring with the pyroglutamic acid amino group, so there are only two free carboxyl groups, statement 5 is correct.

<Checkpoint>[There are a total of two free carboxyl groups in polypeptide **X**](1)</Checkpoint>

Therefore, option C is correct.
根据所给图片信息，可以归类出：在二级质谱中，多肽肽键发生碎裂，含有酰基部分产生酰基正离子的碎片离子称为b离子；含有氨基部分产生氨基正离子的碎片离子称为y离子。

<Checkpoint>[在二级质谱中，多肽肽键发生碎裂，含有酰基部分产生酰基正离子的碎片离子称为b离子；含有氨基部分产生氨基正离子的碎片离子称为y离子。](1)</Checkpoint>

因此，b离子质荷比加17为对应氨基酸分子量，y离子质荷比减一为对应氨基酸分子量。同时，b离子序数增加相当于多肽从N端开始增加氨基酸；y离子序数增加相当于多肽从C端开始增加氨基酸。

<Checkpoint>[b离子质荷比加17为对应氨基酸分子量，y离子质荷比减一为对应氨基酸分子量](1)</Checkpoint>

<Checkpoint>[b离子序数增加相当于多肽从N端开始增加氨基酸；y离子序数增加相当于多肽从C端开始增加氨基酸](1)</Checkpoint>

分析表格数据，y1离子对应氨基酸的分子量为89，为Ala；b1离子对应氨基酸的分子量为129，无对应的氨基酸，应为被修饰的氨基酸，说法6正确。

<Checkpoint>[b1离子对应氨基酸的分子量为129，应为被修饰的氨基酸](1)</Checkpoint>

y2离子对应多肽的分子量为188.1，减去Ala残基的分子量为117.1，为Val；b2离子对应多肽的分子量为186.1，减去被修饰氨基酸残基的分子量为75.1，为Gly；y3离子对应多肽的分子量为245.1，减去y2离子对应多肽的分子量为57，对应氨基酸残基质量，补充一分子水后为75，对应氨基酸质量，为Gly。后续b、y离子采用相同方法进行分析，可以分别得到多肽序列为Mod-Gly-Glu-Asp-Gln-Gly-Val-Ala，其中Mod指代被修饰的氨基酸，被修饰的氨基酸位于N端。从而说法8正确，1错误。

<Checkpoint>[多肽序列为Mod-Gly-Glu-Asp-Gln-Gly-Val-Ala，被修饰的氨基酸位于N端](2)</Checkpoint>

对于多肽**X**的组成，共有$8!/2! = 20160$种可能的序列，说法2正确

<Checkpoint>[多肽**X**的组成，共有$8!/2! = 20160$种可能的序列](1)</Checkpoint>

被修饰的氨基酸经过水解与原氨基酸相同，则是一种可水解的修饰，考虑为酰化修饰；

<Checkpoint>[考虑氨基酸修饰为酰化修饰](1)</Checkpoint>

对比可产生酰化修饰的Ser、Tyr、Thr、Arg、Lys、Cys、Asn/Asp、Gln/Glu，仅有为Gln/Glu时差值为17/18，对应一分子氨或水，则该修饰氨基酸为焦谷氨酸，说法3错误。

<Checkpoint>[被修饰的氨基酸由Gln或Glu产生，修饰为焦谷氨酸](1)</Checkpoint>

焦谷氨酸氨基与侧链羧基酰化成环，N端被封闭，无法通过Edman降解测序，说法7错误。

<Checkpoint>[焦谷氨酸氨基与侧链羧基酰化成环，N端被封闭，无法通过Edman降解测序。](1)</Checkpoint>

多肽**X**中共有C端羧基、一个Asp的侧链羧基、一个Glu的侧链羧基，共三个游离羧基，但Asp的游离羧基与焦谷氨酸氨基成环，因此只有两个游离羧基，说法5正确。

<Checkpoint>[多肽**X**中共两个游离羧基](1)</Checkpoint>

故选项C正确。