Figure 2. Detection limit of mass spectrometry methodology.

1. MALDI‐TOF mass spectrum of trypsin‐digested, synthetic Aβ1‐42 (10 pmol). The Aβ17‐28 [MH]⁺ fragment was detected with the highest intensity. Measurements in reflectron positive mode. The sequence of Aβ1‐42 with the tryptic cleavage sites is indicated at the top.
2. MS/MS spectrum of unlabeled Aβ17‐28 and ¹³C¹⁵N‐Lys Aβ17‐28 synthetic peptides in the 50 fmol range. m/z of precursors ions (m/z 663.34 for Aβ17‐28 [MH]²⁺, m/z 667.36 for ¹³C¹⁵N‐Lys Aβ17‐28 [MH]²⁺) and their derived fragments are indicated as a series of y ions (y4‐y10). The peptides were analyzed in data‐independent acquisition MS^E mode.
3. Decreasing quantities of synthetic ¹³C¹⁵N‐Lys Aβ1‐42 were trypsin‐digested and analyzed by MRM. The intensities of 4 transitions (y10, y9, y8, and y7) were assessed for ¹³C¹⁵N‐Lys Aβ17‐28, showing a detection limit level of 25 amol. Measurements in technical triplicates, normalization to total ion current. Error bars show mean ± SD.
4. Decreasing quantities of synthetic ¹³C¹⁵N‐Lys Aβ1‐42 were spiked into the insoluble fraction of APPtg brains, immunoprecipitated, trypsin‐digested, and analyzed by MRM. Assessment of the intensities of y10, y9, y8, and y7 showed a detection limit level of 50 amol for ¹³C¹⁵N‐Lys Aβ17‐28 and detected an excess of brain‐derived unlabeled Aβ17‐28. Measurements in technical triplicates, normalization to total ion current. Error bars show mean ± SD.

Source data are available online for this figure.