Easy, Robust, and Repeatable Online Acid Cleavage of Proteins in Mobile Phase for Fast Quantitative LC-MS Bottom-Up Protein Analysis─Application for Ricin Detection

Abstract

Sample preparation involving the cleavage of proteins into peptides is the first critical step for successful bottom-up proteomics and protein analyses. Time- and labor-intensiveness are among the bottlenecks of the commonly used methods for protein sample preparation. Here, we report a fast online method for postinjection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics. The chemical cleavage is achieved in 0.1% formic acid within 35 s in a capillary heated to 195 °C installed upstream of the analytical column, enabling the generated peptides to be separated. The peptides generated by the optimized method covered the entire sequence except for one amino acid of trastuzumab used for the method development. The qualitative results are extraordinarily stable, even over a long period of time. Moreover, the method is also suitable for accurate and repeatable quantification. The procedure requires only one manual step, significantly decreasing sample transfer losses. To demonstrate its practical utility, we tested the method for the fast detection of ricin. Ricin can be unambiguously identified from an injection of 10 ng, and the results can be obtained within 7-8 min after receiving a suspicious sample. Because no sophisticated accessories and no additional reagents are needed, the method can be seamlessly transferred to any laboratory for high-throughput proteomic workflows.

Figure 1

Simple apparatus used for the online acid cleavage of proteins in the mobile phase. The final configuration is shown, i.e., without the gradient mixer and with the alumina beads.

Figure 2

Base peak (BP) and total ion current (TIC) chromatograms acquired from trastuzumab cleaved in the reaction capillary at 195 °C.

Figure 3

Effect of cleavage settings in Byonic search on the number of peptide-spectrum matches (PSM) at different temperatures.

Figure 4

Effect of temperature of the reaction capillary (a), flow rate (b), gradient mixer volume (c), and formic acid concentration in the sample (d) on the number of identified unique peptide sequences (uPSs). The effect of reduction of disulfide bonds on uPSs and the portion of those containing Cys (e). Trastuzumab reduced in dithiothreitol (DTT) was prepared and analyzed separately for the non-reduced sample (no DTT) and the sample reduced in 5 mM tris(2-carboxyethyl)phosphine (TCEP 5 mM). Identified uPSs using our method (35 s) and the offline method in 2% formic acid at 108 °C for 2 and 4 h (f).

Figure 5

Number of peptide-spectrum matches (PSM) identified using various cleavage specificity from data obtained using our method (35 s) and the offline method for acid cleavage in 2% formic acid at 108 °C for 2 (2 h) and 4 h (4 h).

Figure 6

Run-to-run repeatability of our method in terms of the number of identified unique peptide sequences (uPSs) across 22 triplicates (a). Two triplicates with a CV greater than 3% are shown as circles. A quantitative Venn diagram showing unique peptide sequences identified repeatedly within triplicate #20 (b). Long-term repeatability of the method in terms of the number of identified uPSs across 9 triplicates measured under identical conditions (c). The time in days, since the first triplicate was analyzed is shown above each subsequent triplicate.

Figure 7

Quantitative repeatability of the most responsive peptides generated via the online acid cleavage (a). The coefficient of variation (CV) for 50 and 100 peptides from light and heavy chains of trastuzumab was calculated from six replicates. Correlation of LC-MS peak areas between replicate 1 and 3 with the worst observed Spearman coefficient (b).

Figure 8

Sequence coverage of ricin chains and the average number of total identified peptides obtained from various injected sample amounts.

Figure 9

Quantitative performance of the three most intense peptides generated from ricin chain A (a–c) and chain B (d–f). Peptides AEAITHLFT and VSILIPIIALMVYRCAPPPSSQ are shared with castor bean agglutinin. Each replicate was considered as an individual point. Dot lines determine bands of 95% confidence.