Mass Spectrum: Videos & Practice Problems

Mass spectrometry analyzes protein structures by generating mass spectra, which plot relative abundance against m/z ratios. Key fragments, known as b ions and y ions, arise from peptide bond cleavage. B ions contain the N-terminal residue and are read left to right, while y ions contain the C-terminal residue and are read right to left. This directional analysis reveals the primary structure of proteins, with y ions typically being more stable and prominent in spectra. Understanding these concepts is crucial for interpreting mass spectrometry data effectively.

Mass Spectrum

A mass spectrum is a graphical representation of mass spectrometry data, plotting relative abundance on the y-axis and the mass-to-charge (m/z) ratio on the x-axis. Each peak in the mass spectrum corresponds to different peptide fragments resulting from the cleavage of an original peptide at specific peptide bonds. Understanding how to analyze a mass spectrum is crucial for revealing the primary structure of proteins, which includes the amino acid composition and sequence from the N-terminal to the C-terminal end.

In a mass spectrum, the largest peaks represent unfragmented peptides, which have the highest m/z ratios and appear furthest to the right. For example, if a pentapeptide (a peptide with five amino acids) does not fragment, it will show a peak corresponding to its total mass. If a peptide bond is cleaved, the resulting fragment will have a lower mass, and the difference in mass between the peaks can be used to identify the amino acid that was removed. This is done by calculating the mass difference (Δmass) and comparing it to known molecular weights of amino acids.

For instance, if the mass difference between two peaks is 99.08 g/mol, it corresponds to the amino acid valine. Similarly, if the difference is 137.18 g/mol, it indicates asparagine. This process continues for each peptide bond cleaved, allowing for the identification of all amino acids in the sequence. The analysis typically proceeds from the right to the left of the spectrum, revealing the sequence from the N-terminal to the C-terminal end.

However, mass spectrometry has limitations, such as the inability to distinguish between leucine and isoleucine, which are isomers with identical masses. This characteristic poses a challenge in accurately identifying these amino acids based solely on mass spectrometry data.

In summary, mass spectrometry is a powerful tool for determining the primary structure of proteins by analyzing mass spectra, identifying amino acids through mass differences, and understanding the fragmentation patterns of peptides.

Use the mass spectrum below to determine the sequence of the peptide.

In peptide sequencing using mass spectrometry, the analysis begins by interpreting the mass spectrum, which plots relative abundance against the mass-to-charge (m/z) ratio. The key to determining the peptide sequence lies in reading the spectrum from right to left, which reveals the sequence from the N-terminal to the C-terminal end of the peptide.

The largest peak on the right corresponds to the intact peptide, while subsequent peaks indicate the presence of specific amino acid residues. For instance, if a peak is associated with a glycine residue, the difference in m/z ratios between peaks can be used to identify the amino acids sequentially. In this case, the first residue identified is glycine (G), followed by valine (V). When encountering leucine (L) or isoleucine (I), it is important to note that mass spectrometry cannot distinguish between these two isomers due to their identical mass, hence they are represented as L/I.

Continuing this process, the sequence is built by identifying each amino acid from the peaks, leading to a sequence that may include multiple residues such as valine, alanine (A), serine (S), aspartic acid (D), tyrosine (Y), and proline (P). The final residue identified will be the C-terminal amino acid, which completes the peptide sequence.

In summary, by systematically analyzing the mass spectrum from right to left, one can accurately deduce the amino acid sequence of the peptide, ensuring to account for any ambiguities with isomeric residues. This method is essential for understanding protein structure and function in biochemistry.

Mass Spectrum

Mass spectrometry is a powerful technique used to analyze the primary structure of proteins by examining the fragments generated during the ionization process. When a protein is ionized, it typically fragments at a single peptide bond, resulting in two main types of ions: b ions and y ions. Understanding these ions is crucial for interpreting mass spectra and determining the sequence of amino acids in a protein.

b ions are characterized by containing the N-terminal amino acid residue of the peptide. These ions are represented in the mass spectrum from left to right. Conversely, y ions contain the C-terminal amino acid residue and are read from right to left. This directional reading aligns with the sequence analysis of the protein, making y ions particularly significant for revealing the amino acid sequence.

To differentiate between b and y ions, one can remember that 'b' is the second letter of the alphabet, indicating that b ions represent the beginning of the protein sequence, while 'y', being the second to last letter, signifies the end of the sequence. This mnemonic aids in recalling that b ions are read from the start of the spectrum and y ions from the end.

For example, consider a pentapeptide undergoing fragmentation. When a peptide bond is cleaved, it generates a b ion that includes the N-terminal residue and a y ion that includes the C-terminal residue. If the peptide bond between two specific amino acids is broken, the resulting b ion might consist of several amino acids, while the y ion could represent a single amino acid. This fragmentation process can be repeated for different peptide bonds, consistently yielding b and y ions that reflect the sequence of the original protein.

Both b and y ions carry a charge and are deflected in an electric field, allowing them to be detected in a mass spectrum. However, y ions are generally more stable and abundant than b ions, often appearing as the most prominent peaks in the spectrum. This stability means that y ions are typically the focus of analysis in mass spectrometry, as they provide clearer insights into the protein's structure.

In practice, mass spectra may display a mixture of b and y ions, but the emphasis is usually placed on y ions due to their higher intensity. As a result, when interpreting mass spectra, it is common to analyze y ions from right to left, which aligns with the sequence of amino acids in the protein. This approach simplifies the process of deducing the protein's primary structure from the mass spectrum.

Upon fragmentation of a peptide bond during mass spectrometry, what ions can be detected on the spectrum?

• A. b ions.
• B. y ions.
• C. b & y ions.
• D. Ions are deflected but not detected.

Answer: (C)

Use the mass spectrum below & the indicated y-ion peaks (red) to reveal the sequence of the peptide.

In peptide sequencing, mass spectrometry is a powerful technique used to determine the order of amino acids in a peptide. The process involves analyzing the mass spectrum, which displays the relative abundance of ions against their mass-to-charge (m/z) ratios. In this context, y ion peaks are particularly important as they provide information about the C-terminal end of the peptide.

To deduce the peptide sequence, one starts from the right side of the mass spectrum, where the peak representing the intact peptide is located. For instance, if the m/z ratio of the intact peptide is 1,298.6, and the adjacent y ion peak is at 1,199.52, the mass difference can be calculated as follows:

$$\text{Mass difference} = 1,298.6 - 1,199.52 = 99.08$$

This mass difference corresponds to the amino acid valine, which has a residue mass of 99.08 g/mol. Thus, the first amino acid in the sequence is valine (V).

Continuing this process, the next peak at 1,142.44 gives a mass difference of:

$$\text{Mass difference} = 1,199.52 - 1,142.44 = 57.08$$

This value matches glycine, indicating that glycine (G) is the next residue. By systematically calculating the mass differences between consecutive y ion peaks, one can identify the following residues:

• Tyrosine (Y) from a mass difference of 163.18
• Valine (V) again from a mass difference of 99.08
• Aspartic acid (D) from a mass difference of 115.08
• Threonine (T) from a mass difference of 101.08
• Glutamine (Q) from a mass difference of 128.14
• Phenylalanine (F) from a mass difference of 147.18
• Tryptophan (W) from a mass difference of 186.54
• Serine (S) from a mass difference of 87.08

By compiling these identified residues, the complete peptide sequence can be constructed. This methodical approach to analyzing mass spectra not only reveals the sequence of the peptide but also enhances understanding of the underlying principles of mass spectrometry in protein analysis.

In your mass-spectrometry of a pure protein with an m/z of 1,582, you found peaks of y ions with the following m/z ratios of 1,582, 1396 and 1283. The mass in Daltons for the possible relevant amino acids are provided: Y (163), N (114), W (186), D (115), G (57), L (113) and M (131). From this data, it is obvious that the C-terminal amino acid residue of the 1,582 fragment is:

• A. G.
• B. L.
• C. N.
• D. W.
• E. Can't be determined.

Answer: (E)

Use the mass spectrum below & the provided chart with amino acid masses to determine the sequence of a hexapeptide (6 amino acid residues). In the mass spectrum, y ion peaks are indicated with 'y' while b ion peaks are indicated with 'b.' The N-terminal residue is given as Leu and the C-terminal residue is given as Lys. Determine the remaining amino acid sequence using either the y ions or the b ions.

In peptide sequencing, mass spectrometry is a powerful tool that allows scientists to determine the sequence of amino acids in a peptide. In this case, we are analyzing a hexapeptide, which consists of six amino acid residues. The N-terminal residue is leucine (L), and the C-terminal residue is lysine (K). The mass spectrum provides peaks corresponding to y ions and b ions, which are crucial for deducing the sequence.

Y ions represent fragments of the peptide that include the C-terminal end, while b ions include the N-terminal end. For a hexapeptide, the unfragmented y ion would be labeled as y₆, but in this spectrum, the highest observed y ion is y₅. This indicates that the y₅ ion is missing the N-terminal leucine, and thus, it contains the last five residues of the peptide.

To determine the sequence, we analyze the differences in mass between the y ion peaks. For instance, the difference between the y₄ and y₅ peaks reveals the mass of the amino acid asparagine (N), which corresponds to a mass of 114.08 Da. Continuing this process, the next differences yield valine (V) with a mass of 99.08 Da, tryptophan (W) with a mass of 186.18 Da, and finally, glycine (G) with a mass of 57.08 Da when subtracting the mass of lysine from the y₂ peak. Thus, the sequence from N-terminal to C-terminal is L-N-V-W-G-K.

Similarly, b ions can also be used to confirm the sequence. The b₂ ion contains the first two residues, and by subtracting the mass of leucine, we find asparagine again. Continuing with the b ions, we find valine, tryptophan, and glycine, confirming the same sequence obtained from the y ions. This illustrates how both y and b ions can be utilized in mass spectrometry to accurately determine and confirm the sequence of peptides.

Here’s what students ask on this topic:

What is a mass spectrum in mass spectrometry?

A mass spectrum is a graphical representation of mass spectrometry data. It plots the relative abundance of detected ions on the y-axis against their mass-to-charge (m/z) ratios on the x-axis. Each peak in the spectrum corresponds to a specific ion, with the height of the peak indicating its relative abundance. By analyzing the differences in m/z ratios between peaks, one can deduce the primary structure of proteins, including the sequence and composition of amino acids. This analysis is crucial for understanding protein fragmentation and identifying specific residues.

How are b ions and y ions different in mass spectrometry?

In mass spectrometry, b ions and y ions are fragments generated from the cleavage of peptide bonds. B ions contain the N-terminal amino acid residue and are read from left to right in the spectrum. In contrast, y ions contain the C-terminal amino acid residue and are read from right to left. Y ions are generally more stable and prominent in the spectrum, making them key for determining the protein sequence. Understanding the distinction between these ions is essential for interpreting mass spectrometry data accurately.

How can mass spectrometry reveal the primary structure of a protein?

Mass spectrometry reveals the primary structure of a protein by analyzing the mass-to-charge (m/z) ratios of peptide fragments. When a protein is ionized and fragmented, it produces a spectrum with peaks corresponding to different fragments. By examining the differences in m/z ratios between these peaks, one can determine the sequence of amino acids. This process involves identifying specific residues based on their unique molecular weights and reading the spectrum from right to left to reveal the sequence from the N-terminal to the C-terminal end.

Why are y ions more prominent in mass spectrometry spectra?

Y ions are more prominent in mass spectrometry spectra because they are more stable than b ions. This stability results in higher intensity and abundance peaks for y ions. Y ions contain the C-terminal amino acid residue and are read from right to left in the spectrum. Their prominence makes them crucial for determining the protein sequence, as they provide clearer and more reliable data compared to the less stable b ions, which tend to fragment further.

What are the limitations of mass spectrometry in differentiating amino acids?

One significant limitation of mass spectrometry is its inability to differentiate between isomeric amino acids, such as leucine and isoleucine. These amino acids have the same molecular mass but differ in their structural arrangement. Since mass spectrometry relies on mass-to-charge (m/z) ratios, it cannot distinguish between molecules with identical masses. This limitation can complicate the analysis of protein sequences, as it introduces ambiguity in identifying specific residues.