Targeted MS/MS-Based MALDI Imaging Using iprm-PASEF

Spatial proteomics has become an important approach for studying the molelccular composition of tissues while preserving their spatial context. Among the available technologies, tryptic peptide MALDI imaging enables fast, multiplexed, label-free, and antibody-independent protein analysis directly from tissue sections. However, confident peptide identification remains one of the major challenges of current MALDI imaging workflows, as most experiments are limited to MS1 acquisition and therefore lack in situ peptide fragmentation and amino acid sequence information. Consequently, peptide identification often relies on complementary (LC-)MS/MS analyses, which do not always enable unambiguous peptide assignment.Recent advances in trapped ion mobility spectrometry (TIMS) and parallel accumulation-serial fragmentation (PASEF) enable multiplexed, in situ, targeted peptide fragmentation during MALDI imaging experiments. By combining an exploratory TIMS-MS1 survey with targeted precursor selection and imaging parallel reaction monitoring (iprm)-PASEF acquisition, reliable peptide identification can be achieved in MALDI imaging.In this chapter, we describe a detailed protocol for targeted tryptic peptide MS/MS-based MALDI imaging using iprm-PASEF on the timsTOF fleX mass spectrometer. The workflow consists of tissue preparation with in situ protein digestion, followed by exploratory MALDI TIMS-MS1 imaging, precursor list generation, iprm-PASEF acquisition, and subsequent peptide identification. In addition, practical considerations for instrumental setup, precursor selection, and data analysis are discussed to facilitate implementation of the workflow for spatial proteomics applications.