Proteomics 2016, 16, 1695–1706 1695 DOI 10.1002/pmic.201500530 RESEARCH ARTICLE A broad-based study on hyphenating new ionization technologies with MS/MS for PTMs and tissue characterization Darrell D. Marshall 1 , Ellen D. Inutan 1 , Beixi Wang 1 , Chih-Wei Liu 1 , Shameemah Thawoos 1 , James Wager-Miller 2 , Ken Mackie 2 and Sarah Trimpin 1,3 1 Department of Chemistry, Wayne State University, Detroit, MI, USA 2 Department of Psychological & Brain Sciences, Indiana University, Bloomington, IN, USA 3 Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI, USA Received: December 15, 2015 Revised: March 27, 2016 Accepted: April 11, 2016 Matrix-assisted ionization (MAI) is a newly discovered method for converting compounds from the solid phase to gas-phase ions having charge states similar to electrospray ionization (ESI), but without the need for high-energy sources such as lasers or high voltage. Laserspray ioniza- tion (LSI) is a subset of MAI that uses a laser to provide high spatial resolution analyses, but the laser is not directly involved in the ionization process. These methods produce multiply-charged analyte ions that are useful for characterizing compounds directly from surfaces using advanced characterization technologies. Because the multiply-charged ions originate from charged ma- trix clusters, efficient desolvation of the matrix is a prerequisite. Here, we report on the utility of collision-induced dissociation (CID) and electron transfer dissociation (ETD) coupled to mass spectrometry using several MAI and LSI matrices for peptide and protein characterization employing mass spectrometers from two manufacturers. The information obtained is similar to that using ESI for most analyses and superior to matrix-assisted laser desorption/ionization (MALDI) as is shown for intact proteins and protein digests directly from mouse brain tissue sections. The ionization processes are soft so that posttranslational modification (e.g. phosphorylation) sites are readily determined. Instances where ETD or CID in conjunction with MAI failed are attributed to lack of desolvation of charged matrix:analyte particles. Keywords: Bottom-up proteomics / Electron transfer dissociation / Intact proteins / Matrix- assisted ionization / Posttranslational modifications / Technology  Additional supporting information may be found in the online version of this article at the publisher’s web-site Correspondence: Dr. Sarah Trimpin, Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, USA E-mail: strimpin@chem.wayne.edu Fax: +1-313-577-8822 Abbreviations: ACTH, adrenocorticotropic hormone; 2,5-DHAP, 2,5-dihydroxyacetophenone; ETD, electron transfer dissociation; FA, formic acid; IMS, ion mobility spectrometry; LSI, laserspray ionization; MAI, matrix-assisted ionization; MAIV, MAI vacuum; MW, molecular weight; 3-NBN, 3-nitrobenzonitrile; SAI, solvent- assisted ionization; PTM’s, posttranslational modifications 1 Introduction Matrix-assisted ionization (MAI) processes for use in mass spectrometry (MS) have been reported to depend on a com- bination of temperature and pressure rather than lasers or other high-energy means to initiate ionization [1]. These new methods are sensitive, applicable for low- and high-mass compounds using matrices that are solids or solutions [2–7]. Laserspray ionization (LSI) [2, 4], a MAI method for rapid and high spatial resolution analyses and imaging applications op- erates from atmospheric pressure or vacuum [8–12]. MAI Colour Online: See the article online to view Figs. 2–5 in colour. C  2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com