{"id":8449,"date":"2024-01-02T07:59:31","date_gmt":"2024-01-02T07:59:31","guid":{"rendered":"https:\/\/chemistryscore.com\/?p=8449---42965e94-5375-4fd3-a79c-85bcb7a99bd7"},"modified":"2024-01-02T07:59:31","modified_gmt":"2024-01-02T07:59:31","slug":"protein-sequencing-process","status":"publish","type":"post","link":"https:\/\/chemistryscore.com\/protein-sequencing-process\/","title":{"rendered":"Protein Sequencing Process"},"content":{"rendered":"\t\t
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The protein sequencing process describes the steps that need to be taken in order to determine the amino acid sequence of a protein.<\/p>

However, in order to begin, you must take some preliminary steps to ensure that your protein is ready to be sequenced:<\/p>

  1. You must have a\u00a0purified protein<\/a><\/li><\/ol>

    After you confirmed or purified your protein, you can then move onto the actual sequencing process.<\/p>

    Step 1: Separate protein subunits<\/h2>

    What this means:<\/strong>\u00a0A protein can consist of 2 or more polypeptide chains that are linked together by disulfide bonds (see graphic below). For sequencing to properly work, you must break all protein subunits into individual strands.<\/p>

    N-terminal analysis of your protein provides information on the different types of subunits.<\/p>

    Since each polypeptide chain has an N-terminus (also known as the \u201cend group\u201d of a protein) it is possible to use a fluorescent compound such as\u00a05-dimethylamino-1-naphthalenesulfonyl chloride<\/strong>\u00a0(aka \u201cdansyl chloride\u201d) to cleave to the primary amine (N-terminal of a protein is the amine group)<\/p>

    Quick Recap:\u00a0N-terminal vs C-terminal of amino acids<\/a><\/p>

    So What Does Dansyl Chloride Do?<\/h3>

    What dansyl chloride does is it cleaves or attaches itself onto\u00a0the n-terminus of an amino acid, thus fluorescing the amino acid.<\/p>

    Acid hydrolysis is then used to separate the dansylated amino acid from free amino acids as shown below:<\/p>

    \"\"<\/p>

    Chromatography is used to compare the fluoresced amino acid to a known standard to determine the amino acid.<\/p>

    Step 2: Produce Smaller Peptide Fragments<\/h2>

    Remember, protein sequences can be very long!<\/p>

    In fact there are 20n<\/sup>\u00a0sequence combinations possible (n = # of amino acid residues)<\/p>

    If the polypeptides that are longer than 40 to 100 residues cannot be sequenced directly.<\/p>

    So to remedy this situation, you can produce smaller peptide fragments to work with via chemical or enzymatic hydrolysis<\/p>

    Chemical Cleavage:<\/h3>

    CNBr (cyanogen bromide) cleaves onto the methionine (Met) residues of an amino acid.<\/p>

    Enzymatic Cleavage:<\/h3>

    Enzyme cleavage can be tricky because various enzymes cleave differently.<\/p>

    There are:<\/p>

    Endopeptidases:<\/h4>

    Endopeptidases are enzymes that cleave to internal peptide bonds and hydrolyze them.<\/p>

    Exopeptidases:<\/h4>

    Exopeptidases are enzymes that cleave to terminal C or N residues of amino acids in order to fragment polypeptides.<\/p>

    Proteases:<\/h4>

    Proteases act as both exopeptidases and endopeptidases.<\/p>

    However, these enzymes have specific side chain requirements in order for cleavage to occur. This means that the identity of the amino acid\u00a0before<\/em>\u00a0the one being cleaved matters.<\/p>

    Step 3: Sequence the protein fragment<\/h2>

    Once you have your fragment. You are ready to sequence your protein. Two common methods are\u00a0Edman Degradation<\/a>\u00a0and Tandem Mass Spec<\/p>

    Step 4: Repeat steps 2 and steps 3 using a different cleavage\/hydrolysis method<\/h2>

    A different cleavage method will give you different peptide fragments that will overlap your initial sequencing. At the end, you will put together your overlapping sequences to get your final sequence.<\/p>

    \"\"\u00a0Source: Voet, Voet, and Pratt<\/p>

    Step 5: Assemble the overlapping fragments for the full protein sequence<\/h2>

    As stated, different fragments derived from different cleavage mechanisms yield various sequences. At the end, the sequences are overlapped to obtain the full ordered sequence.<\/p>\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<\/div>\n\t\t","protected":false},"excerpt":{"rendered":"

    The protein sequencing process describes the steps that need to be taken in order to determine the amino acid sequence of a protein. However, in order to begin, you must take some preliminary steps to ensure that your protein is ready to be sequenced: You must have a purified protein After you confirmed or purified your […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"ocean_post_layout":"","ocean_both_sidebars_style":"","ocean_both_sidebars_content_width":0,"ocean_both_sidebars_sidebars_width":0,"ocean_sidebar":"0","ocean_second_sidebar":"0","ocean_disable_margins":"enable","ocean_add_body_class":"","ocean_shortcode_before_top_bar":"","ocean_shortcode_after_top_bar":"","ocean_shortcode_before_header":"","ocean_shortcode_after_header":"","ocean_has_shortcode":"","ocean_shortcode_after_title":"","ocean_shortcode_before_footer_widgets":"","ocean_shortcode_after_footer_widgets":"","ocean_shortcode_before_footer_bottom":"","ocean_shortcode_after_footer_bottom":"","ocean_display_top_bar":"default","ocean_display_header":"default","ocean_header_style":"","ocean_center_header_left_menu":"0","ocean_custom_header_template":"0","ocean_custom_logo":0,"ocean_custom_retina_logo":0,"ocean_custom_logo_max_width":0,"ocean_custom_logo_tablet_max_width":0,"ocean_custom_logo_mobile_max_width":0,"ocean_custom_logo_max_height":0,"ocean_custom_logo_tablet_max_height":0,"ocean_custom_logo_mobile_max_height":0,"ocean_header_custom_menu":"0","ocean_menu_typo_font_family":"0","ocean_menu_typo_font_subset":"","ocean_menu_typo_font_size":0,"ocean_menu_typo_font_size_tablet":0,"ocean_menu_typo_font_size_mobile":0,"ocean_menu_typo_font_size_unit":"px","ocean_menu_typo_font_weight":"","ocean_menu_typo_font_weight_tablet":"","ocean_menu_typo_font_weight_mobile":"","ocean_menu_typo_transform":"","ocean_menu_typo_transform_tablet":"","ocean_menu_typo_transform_mobile":"","ocean_menu_typo_line_height":0,"ocean_menu_typo_line_height_tablet":0,"ocean_menu_typo_line_height_mobile":0,"ocean_menu_typo_line_height_unit":"","ocean_menu_typo_spacing":0,"ocean_menu_typo_spacing_tablet":0,"ocean_menu_typo_spacing_mobile":0,"ocean_menu_typo_spacing_unit":"","ocean_menu_link_color":"","ocean_menu_link_color_hover":"","ocean_menu_link_color_active":"","ocean_menu_link_background":"","ocean_menu_link_hover_background":"","ocean_menu_link_active_background":"","ocean_menu_social_links_bg":"","ocean_menu_social_hover_links_bg":"","ocean_menu_social_links_color":"","ocean_menu_social_hover_links_color":"","ocean_disable_title":"default","ocean_disable_heading":"on","ocean_post_title":"","ocean_post_subheading":"","ocean_post_title_style":"","ocean_post_title_background_color":"","ocean_post_title_background":0,"ocean_post_title_bg_image_position":"","ocean_post_title_bg_image_attachment":"","ocean_post_title_bg_image_repeat":"","ocean_post_title_bg_image_size":"","ocean_post_title_height":0,"ocean_post_title_bg_overlay":0.5,"ocean_post_title_bg_overlay_color":"","ocean_disable_breadcrumbs":"default","ocean_breadcrumbs_color":"","ocean_breadcrumbs_separator_color":"","ocean_breadcrumbs_links_color":"","ocean_breadcrumbs_links_hover_color":"","ocean_display_footer_widgets":"default","ocean_display_footer_bottom":"default","ocean_custom_footer_template":"0","_jetpack_memberships_contains_paid_content":false,"ocean_post_oembed":"","ocean_post_self_hosted_media":"","ocean_post_video_embed":"","ocean_link_format":"","ocean_link_format_target":"self","ocean_quote_format":"","ocean_quote_format_link":"post","ocean_gallery_link_images":"off","ocean_gallery_id":[],"footnotes":""},"categories":[6,28],"tags":[],"jetpack_sharing_enabled":true,"jetpack_featured_media_url":"","yoast_head":"\nProtein Sequencing Process - ChemistryScore<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/chemistryscore.com\/protein-sequencing-process\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Protein Sequencing Process - ChemistryScore\" \/>\n<meta property=\"og:description\" content=\"The protein sequencing process describes the steps that need to be taken in order to determine the amino acid sequence of a protein. 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