Extending the Mannose 6-Phosphate Glycoproteome by High Resolution/Accuracy Mass Spectrometry Analysis of Control and Acid Phosphatase 5-Deficient Mice

Abstract

In mammals, most newly synthesized lumenal lysosomal proteins are delivered to the lysosome by the mannose 6-phosphate (Man6P) targeting pathway. Man6P -containing proteins can be affinity-purified and characterized using proteomic approaches, and such studies have led to the discovery of new lysosomal proteins and associated human disease genes. One limitation to this approach is that in most cell types the Man6P modification is rapidly removed by acid phosphatase 5 (ACP5) after proteins are targeted to the lysosome, and thus, some lysosomal proteins may escape detection. In this study, we have extended the analysis of the lysosomal proteome using high resolution/accuracy mass spectrometry to identify and quantify proteins in a combined analysis of control and ACP5-deficient mice. To identify Man6P glycoproteins with limited tissue distribution, we analyzed multiple tissues and used statistical approaches to identify proteins that are purified with high specificity. In addition to 68 known Man6P glycoproteins, 165 other murine proteins were identified that may contain Man6P and may thus represent novel lysosomal residents. For four of these lysosomal candidates, (lactoperoxidase, phospholipase D family member 3, ribonuclease 6, and serum amyloid P component), we demonstrate lysosomal residence based on the colocalization of fluorescent fusion proteins with a lysosomal marker. Molecular & Cellular

Analysis of novel endosome-to-Golgi retrieval genes reveals a role for PLD3 in regulating endosomal protein sorting and amyloid precursor protein processing

“…The localisation of PLD3 to lysosomes is consistent with other reports describing the localisation of PLD3 [23]. We, however, observed a significant pool of the PLD3 protein in structures that were positive for retromer proteins (i.e.…”

Proteomic Analysis of Brain and Cerebrospinal Fluid from the Three Major Forms of Neuronal Ceroid Lipofuscinosis Reveals Potential Biomarkers

“…Preparations of purified Man6 P glycoproteins and their corresponding mock eluates were analyzed using a Thermo Orbitrap Velos and analyzed, as previously described in detail. 16 iTRAQ 8-plex labeling was conducted using manufacturer's (Thermo Scientific) methods. Samples were fractionated by alkaline RP-HPLC, and ∼20 fractions were analyzed in duplicate by HCD on a Thermo Orbitrap Velos using column and gradient conditions as described.…”

Using whole‐exome sequencing to investigate the genetic bases of lysosomal storage diseases of unknown etiology

“…Table S1). This list was based on multiple sources including: (1) proteomic analyses of lysosomes and lysosomal proteins (Bagshaw et al., ; Chapel et al., ; Schroder et al., ), (2) genes involved in lysosomal related pathways (Di Fruscio et al., ), (3) proteins transcribed by TFEB, a master gene for lysosomal biogenesis (Palmieri et al., ), (4) glycoproteins that contain the mannose 6‐phosphate lysosomal targeting modification (Sleat et al., ; Sleat et al., ), and (5) global protein subcellular localization studies (Jadot et al., ).…”