{"id":8966,"date":"2016-11-04T11:00:06","date_gmt":"2016-11-04T11:00:06","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/proteomics\/?p=8966"},"modified":"2016-11-04T11:00:06","modified_gmt":"2016-11-04T11:00:06","slug":"why-is-osteocalcin-undetected-by-mass-spectrometry","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/proteomics\/why-is-osteocalcin-undetected-by-mass-spectrometry\/","title":{"rendered":"Why Is Osteocalcin Undetected by Mass Spectrometry?"},"content":{"rendered":"<p><img loading=\"lazy\" decoding=\"async\" src=\"http:\/\/www.thermofisher.com\/blog\/proteomics\/wp-content\/uploads\/sites\/2\/2016\/11\/shutterstock_262384457.jpg\" style=\"float: left;margin: 5px\" alt=\"Digital composite of highlighted bones of man about to race. Image: ESB Professional\/Shutterstock.com.\" width=\"330\" height=\"220\" \/>Osteocalcin (OC) is an important protein in the extracellular matrix of bone that controls hydroxyapatite crystal properties. It also&nbsp;plays a role in whole-bone mechanics because of its interaction with both hydroxyapatite and bone collagen I. OC is of particular interest in human and&nbsp;model organism studies due to its <span class=\"thread\">roles within bone and<\/span>&nbsp;as a hormone promoting insulin production. However, OC is&nbsp;difficult to detect using high-throughput tandem mass spectrometry (MS\/MS) proteomic approaches from bone protein extracts. Researchers predominantly use&nbsp;non-MS immunological methods to detect OC.&nbsp;Cleland et al. (2016) sought to&nbsp;determine why OC goes undetected.<sup>1<\/sup><\/p>\n<p>The investigators used bovine OC isolated from&nbsp;<em>Bos taurus <\/em>bone. They prepared samples for analysis as OC peptides and intact OCs. They also prepared a sample&nbsp;with bovine collagen I and OC in a 100:1 ratio, to reflect a similar ratio of collagen to OC in bone and demonstrate the effect of collagen I on OC detection by MS. Cleland et al. separated all three OC preparations for 75 minutes at increasing gradients and then characterized the eluted peptides&nbsp;in positive mode on an&nbsp;<a href=\"https:\/\/www.thermofisher.com\/order\/catalog\/product\/IQLAAEGAAPFADBMAOK\" target=\"_blank\">LTQ Orbitrap XL mass spectrometer<\/a> <span class=\"thread\"><\/span>(Thermo Scientific). They fragmented the three&nbsp;most abundant peaks using h<span>igher energy collision dissociation<\/span>&nbsp;(HCD). To identify OC peaks, Cleland et al. <span class=\"thread\">converted&nbsp;<\/span>raw files to&nbsp;peak lists and searched against Swiss-Prot and a decoy database&nbsp;using Mascot 2.3. Finally, the investigators performed&nbsp;in&nbsp;silico<em>&nbsp;<\/em>digestion of <em>B. taurus <\/em>OC. They extracted averaged MS\/MS spectra&nbsp;corresponding to these peaks.<\/p>\n<p>For intact OC,&nbsp;Cleland et al. detected&nbsp;proteoforms with up to three carboxylations. They also identified&nbsp;chromium-adducted forms with up to three carboxylations. They&nbsp;found fully carboxylated <em>B. taurus <\/em>OC&nbsp;with hydroxyproline. They also found&nbsp;doubly, singly and uncarboxylated proteoforms.&nbsp;The chromium-adducted forms showed more consistent carboxylation levels, which the investigators&nbsp;suggest indicates that the chromium <span class=\"thread\">stabilizes<\/span>&nbsp;the OC carboxylation, and that some of the carboxylation variation detected is the result of decarboxylation during or before their analysis. Further, they found&nbsp;extensive overalkylation of the cysteine-free N-terminal peptide&nbsp;and the dicysteine middle OC peptide. The investigators suggest that this makes an added N-terminal carbamidomethylation (CAM) modification in Mascot critical to detect these peptides.<\/p>\n<p>Cleland et al. note the following hindrances to OC detection by MS:<\/p>\n<ol>\n<li>\n<p>Gamma carboxylation of one or more glutamic acids<\/p>\n<\/li>\n<li>\n<p>OC acidity: gamma carboxylation of glutamic acid results in neutral loss after fragmentation<\/p>\n<\/li>\n<\/ol>\n<p>In positive ionization mode, acidic peptides show reduced ion signals&nbsp;compared to relatively<span class=\"thread\">&nbsp;<\/span>basic ones, such as&nbsp;those from collagen I. In the samples prepared with&nbsp;bovine collagen I, the investigators could not detect OC.<\/p>\n<p>In conclusion,&nbsp;Cleland et al. recommend that researchers do not use HCD fragmentation. They should instead consider alternative fragmentation methodology&nbsp;and\/or additional search strategies.<\/p>\n<p><strong>Reference<\/strong><br \/>1. Cleland, T.P. (2016) &ldquo;<a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/27470908\" target=\"_blank\">Influence of carboxylation on osteocalcin detection by mass spectrometry<\/a>,&rdquo; Rapid Communications in Mass Spectrometry,&nbsp;<span>30(19) (pp. 2109&ndash;2115), doi: 10.1002\/rcm.7692.<\/span><\/p>\n<p><i>Post Author: Miriam Pollak. Miriam a Nutritionist specialising in women&#8217;s health and works from her Bondi Beach clinic. She is also currently completing her Masters by research in nutrition.<\/p>\n<p>Prior to this, Miriam majored in neuroscience as an undergraduate before completing a post graduate degree in science communication. She spent over a decade working in science communication and medical research, collaborating with some of the best oncologists and researchers in Australia and the U.S.<\/i><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Osteocalcin (OC) is an important protein in the extracellular matrix of bone that controls hydroxyapatite crystal properties. It also&nbsp;plays a role in whole-bone mechanics because of its interaction with both hydroxyapatite and bone collagen I. OC is of particular interest in human and&nbsp;model organism studies due to its roles within bone and&nbsp;as a hormone promoting<\/p>\n","protected":false},"author":7,"featured_media":8965,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[309],"tags":[169],"division":[],"class_list":{"0":"post-8966","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-musculoskeletal","8":"tag-research-news","9":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"<!-- 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