Differentiation of Isomeric TAT1-CARNOSINE Peptides by Energy-Resolved Mass Spectrometry and Principal Component Analysis

Abstract

L-carnosine (Car) is an endogenous dipeptide with significant potential in drug discovery for neurodegenerative diseases, while TAT1, a small arginine-rich peptide derived from the HIV-1 trans-activator protein (TAT), is known to stimulate proteasome activity. In this study, three isomeric peptides were synthesised by incorporating the Car moiety at the N-terminus, C-terminus, or central position of the TAT1 sequence. To differentiate these isomers, high-resolution and energy-resolved CID MS/MS experiments were conducted. The resulting MS/MS spectra showed a high degree of similarity among the peptides, predominantly characterised by fragment ion peaks arising from arginine-specific neutral losses. Energetic analysis was similarly inconclusive in resolving the isomers. However, Principal Component Analysis (PCA) enabled clear differentiation of the three peptides by considering the entire MS/MS spectra rather than focusing solely on precursor ion intensities or major fragment peaks. PCA loadings revealed distinct fragment ions for each peptide, albeit with lower intensities, providing insights into consecutive fragmentation patterns. Some of these specific peaks could also be attributed to scrambling during fragmentation. These results demonstrate the potential of PCA as a simple chemometric tool for semi-automated peak identification in complex MS/MS spectra.

Keywords: MS/MS; Principal Component Analysis; TAT1; carnosine; energy-resolved mass spectrometry; high-resolution mass spectrometry; isomeric peptides.

Figure 1

Amino acid sequences of carnosine (Car) and TAT1, along with the three isomeric peptides studied, are shown. The colour of carnosine indicates its position within the peptide: in Car-TAT1, carnosine (blue) is bound to the N-terminus; in TAT1-Car, carnosine (green) is bound to the C-terminus; and in T-Car-T, carnosine (red) is located in the middle of the TAT1 amino acid sequence.

Figure 2

High-resolution MS/MS spectrum of Car-TAT1 from 800 to 900 m/z. Acceleration voltage: 36 V. The fragment ions are due to consecutive neutral losses, and they do not provide information on the position of carnosine. This part of the MS/MS spectrum is similar to the three isomeric peptides. The b-type conventional fragment and its consecutive neutral losses are highlighted in red, while the remaining fragments (in black) correspond to non-conventional fragments related to the neutral losses of arginine.

Figure 3

Survival Yield (SY) curve of the double protonated isomeric peptides: Car-TAT1 (blue); TAT1-Car (red); and T-Car-T (green). SY was calculated from high-resolution MS/MS spectra performed at acceleration voltages ranging from 20 V to 42 V. The SY curves are similar for the three peptides.

Figure 4

Principal Component Analysis (PCA) performed on the high-resolution MS/MS spectra of T-Car-T, TAT1-Car, and Car-TAT1 in the m/z range of 100–900. (a) Score plot of the two first principal components (the explained variance for PC1 is 52.05% and 47.95% for PC2). (b) Loading plot of PC1 vs. PC2. Each point (in blue) shows the loading of a mass peak (m/z) for PC1 (in x-axis) and PC2 (in y-axis).

Figure 5

Breakdown curves of the doubly protonated isomeric peptides: Car-TAT1 (blue); TAT1-Car (red); and T-Car-T (green), corresponding to the key fragment ions peak identified as the most significant loadings. The high-resolution MS/MS spectra were acquired at acceleration voltages ranging from 20 V to 42 V.

Figure 6

Principal Component Analysis (PCA) performed on the high-resolution MS/MS spectra of T-Car-T, TAT1-Car, and Car-TAT1 in the reduced m/z range of 600–800. (a) Score plot of the first two principal components, with PC1 explaining 89.97% of the variance and PC2 explaining 10.03%. (b) Loading plot of PC1 versus PC2. Each blue point represents the loading of a mass peak (m/z), with PC1 shown on the x-axis and PC2 on the y-axis.

Figure 7

Breakdown curves of the doubly protonated isomeric peptides: Car-TAT1 (blue); TAT1-Car (red); and T-Car-T (green). The curves correspond to fragment ions identified as characteristic of Car-TAT1 based on the loadings plot. High-resolution MS/MS spectra were acquired at acceleration voltages ranging from 20 V to 42 V.

Figure 8

Extract from the high-resolution MS/MS spectrum of the Car-TAT1 peptide, covering the m/z range from 700 to 800. Ions derived from consecutive neutral losses of [y₁₂]²⁺ are highlighted, although [y₁₂]²⁺ is hardly detected.

Figure 9

Breakdown curves of the doubly protonated isomeric peptides: Car-TAT1 (blue); TAT1-Car (red); and T-Car-T (green). The curves correspond to fragment ions identified as characteristic of T-Car-T based on the loadings plot. High-resolution MS/MS spectra were acquired at acceleration voltages ranging from 20 V to 42 V.

Figure 10

Extract from the high-resolution MS/MS spectrum of the T-Car-T peptide, covering the m/z range from 550 to 800. Ions derived from scrambling and consecutive neutral losses are highlighted.

Figure 11

Breakdown curves of the doubly protonated isomeric peptides: Car-TAT1 (blue); TAT1-Car (red); and T-Car-T (green). The curves correspond to fragment ions identified as characteristic of TAT1-Car based on the loadings plot. High-resolution MS/MS spectra were acquired at acceleration voltages ranging from 20 V to 42 V.

Figure 12

Extract from the high-resolution MS/MS spectrum of the TAT1-Car peptide, covering the m/z range from 360 to 800. Ions derived from conventional fragmentation are highlighted.