利用叠氮丙氨酸标记和流式细胞分析测定细胞周期中蛋白的合成

Koshi   Imami; Tomoharu  Yasuda

doi:10.21769/BioProtoc.3215

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Peer-reviewed

Measuring Protein Synthesis during Cell Cycle by Azidohomoalanine (AHA) Labeling and Flow Cytometric Analysis

利用叠氮丙氨酸标记和流式细胞分析测定细胞周期中蛋白的合成

KI Koshi Imami email

TY Tomoharu Yasuda

发布: 2019年04月20日第9卷第8期 DOI: 10.21769/BioProtoc.3215 浏览次数: 8620

评审: Gal HaimovichAlexandros C KokotosVarpu Marjomaki

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Oct 2018

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Abstract

Protein synthesis is one of the most fundamental biological processes to maintain cellular proteostasis. Azidohomoalaine (AHA) is a non-radioactive and “clickable” amino acid analog of methionine which can be incorporated into newly synthesized proteins. Thus, AHA-labeled nascent proteins can be detected and quantified through fluorescent labeling by "click" chemistry. Here we describe a protocol to measure protein synthesis by AHA labeling and flow cytometry. Taking advantage of gating different cell populations, we provide a typical example of the flow cytometric-based analysis of protein synthesis during the cell cycle. While we used mouse B cells in this protocol this method can be readily applied to any cell types and organisms.

Keywords: Protein synthesis (蛋白合成)

Translation (翻译)

AHA (叠氮丙氨酸)

Click chemistry (点击化学)

Flow cytometry (流式细胞术)

Cell cycle (细胞周期)

Mitosis (有丝分裂)

Non-radioactive amino acid (非放射性氨基酸)

Background

Traditionally measurement of protein synthesis is performed by pulse labeling of translation products using radiolabeled amino acids such as [³⁵S]methionine and [³⁵S]cysteine but the use of radioactive materials is the major disadvantage. Recent advance in bio-orthogonal chemical reporters such as azides and alkynes allows us to quantitatively monitor and track biomolecules (proteins, lipids or nucleic acids) through click chemistry (Dieterich, 2010). Azidohomoalaine (AHA) (Figure 1) is a methionine analog that contains an azide moiety and is incorporated into newly synthesized proteins. Thus, nascent proteins can be detected through "click" reaction between AHA (azido) and a fluorescent alkyne (Dieterich, 2010).

Figure 1. Chemical structures of L-methionine and L-azidohomoalaine (AHA)

Measuring protein synthesis during the cell cycle in mammalian cells has been challenging. To do this, cell synchronization in specific phases of the cell cycle is necessary but the use of drugs such as nocodazole was shown to affect translation (Coldwell et al., 2013; Shuda et al., 2015). To measure protein synthesis without drug treatment, here we used metabolic pulse labeling with AHA in a flow cytometric assay (Kiick et al., 2002; Shuda et al., 2015). In combination with AHA, we used anti-phospho-Histone H3 (Ser10) antibody as a mitotic marker to monitor mitotic translation in mouse B lymphoma cells (19DN) (Sander et al., 2015).

Materials and Reagents

Pipette tips (Eppendorf, epT.I.P.S.)
6-well plate (Thermo Fisher Scientific, BioLite 6-well Multidish, catalog number: 130184)
96-well U- or V-bottom cell culture plate (Corning, catalog number: 6902D09 or 6928A17)
5 ml round bottom polystyrene FACS tube (Falcon, catalog number: 38007)
L-Azidohomoalanine (AHA) (Anaspec, catalog number: AS-63669, 500 mM stock solution in deionized water, keep at -20 °C up to at least a few months)
Methionine-free medium such as Dulbecco’s Modified Eagle’s Medium–high glucose With 4,500 mg/L glucose and sodium bicarbonate, without L-methionine, L-cystine and L-glutamine, liquid, sterile-filtered, suitable for cell culture (Merk, catalog number: D0422)
Fetal bovine serum, South America origin, dialyzed, sterile filtered (Pan Biotech, catalog number: P30-2102)
L-Methionine (Merck, catalog number: M9625, make 300 mM stock solution in deionized water, keep at -20 °C up to at least a few months)
L-Cystine dihydrochloride (Merck, catalog number: C2526, prepare fresh 200 mM (1,000x for DMEM) solution in deionized water)
GlutaMAX (Thermo Fisher Scientific, catalog number: 35050061, 200 mM stock solution)
D-PBS (1x), no calcium chloride, no magnesium chloride, sterile-filtered (Thermo Scientific Fisher, catalog number: 14190144)
Alexa Fluor488 alkyne (Thermo Fisher Scientific, catalog number: A10267)
Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit mAb (Alexa Fluor 647 Conjugate) (Cell Signaling Technology, catalog number: 3458S)
(+)-Sodium L-ascorbate (Merck, catalog number: A7631)
Copper(II)-Sulphate (CuSO₄) (Baseclick, catalog number: BCMI-004-50)
Paraformaldehyde (PFA) (Roth, 335)
Saponin (Sigma-Aldrich, catalog number: 47036)
(Optional) Triton X-100 (Sigma-Aldrich, catalog number: T9284)
Bovine serum albumin (BSA) (Roth, 8076)
DMSO (Merck, catalog number: D8418)
EDTA (Merck, catalog number: EDS)
NaN₃ (Merck, catalog number: 71289)
Cycloheximide (Santa Cruz Biotechnology, catalog number: sc-3508)
Cell culture medium for AHA labeling (w/o methionine or AHA) (see Recipes)
Washing solution (see Recipes)
Fixing solution (see Recipes)
Permeabilization solution (see Recipes)
FACS buffer (see Recipes)

Equipment

(Optional) Multi channel pipette (Eppendorf, catalog number: 3122 000.043 [10-100 μl])
CO₂ Incubator (Thermo Fisher Scientific, catalog number: 51026331)
Refrigerated centrifuge (Eppendorf, models: 5418 R [fixed angle for tubes], 5804 R [swing out for 96 well plates])
Flow cytometer (FACS Aria instrument) (BD Biosciences)

Software

Analysis Software: FlowJo

Procedure

English

中文翻译

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如何引用

Imami, K. and Yasuda, T. (2019). Measuring Protein Synthesis during Cell Cycle by Azidohomoalanine (AHA) Labeling and Flow Cytometric Analysis. Bio-protocol 9(8): e3215. DOI: 10.21769/BioProtoc.3215.