蝙蝠俠對超人：正義曙光
班·艾佛列克、亨利·卡維爾

Date: 20160327
Version: 1

劇情：
故事敘述出自高譚市的治安守護者「蝙蝠俠」將要前往大都會挑戰最受崇敬的現代救世主「超人」。當雙方處於交戰狀態時，一個新的威脅「毀滅日」將使城市混亂，讓人們陷入更大的危機之中。

感想：
1. 我認為主要是劇情片，輔以動作片加成，還算一部賞心悅目的影片，所以如果你想看動作片的話，可能會有點失望，裡面劇情偏多，就不知道美國隊長3是否也是這樣？畢竟內戰也是一個沉重的話題，但漫威一向是爽片的代表，所以不清楚美國隊長3是如何呈現。

2. 稻草人的恐懼藥劑實在是太厲害了，連外星人都有效，太厲害了。

3. 蝙蝠俠打超人那一幕，前面打得很精彩，結果後面一句"瑪莎"，就結束了，雖然這是為了接續後面的劇情，但實在是轉的有點硬，有點虎頭蛇尾的感覺。

4. 其中有一幕是閃電俠，從未來跑回來說"露薏絲·蓮恩"是關鍵，我猜測是在說不義聯盟的故事，小丑差掉超人的女朋友然後，超人就動手殺了小丑，並建立集權政府的故事，我還蠻喜歡不義聯盟的故事，希望之後有機會拍出來。

5. 神力女超人的部分，我覺得除了最後一場戰鬥出來秀一下之外，其他部分神力女超人就向路人一樣路過而已，不過她的出場曲真的不錯聽。

6. 最後，反派的部分，雷克斯·路瑟和末日實在是有夠掉漆的，雷克斯·路瑟可是反派頭目，一下子就去監獄了，然後末日可是幹掉過超人的，結果被三巨頭幹掉，真是太弱了。

7. 為啥外星人的太空船是用指紋辨識啊？這太奇怪了吧？有這麼簡單就可以進去，政府是在耍甚麼白癡嗎？

小說背景設定-3

Date: 20160327
Version: 1

無限恐怖，在經歷過劇情後會得到支線劇情和點數，以兌換各種能力，但無限恐怖之主旨，不就是利用恐懼來激發人的潛能來打開基因鎖，那為什麼"能"兌換能力呢？一旦兌換能力，潛意識就會認為有了依杖，這可能會阻礙無限恐怖之目的，我們需要的是激發潛能，不是需要你兌換各種能力。

所以，如果取消能力這一項的話，可能有幾種方案：
1. 完成劇情後不會獲得支線劇情和點數，這樣就沒辦法兌換能力
2. 主神空間並不提供兌換能力的服務
3. 回到主神空間後，會移除身上所有的能力

但換個角度來想，如果兌換能力後，可以接受更高的恐懼，這樣是否可以更激發人的潛能呢？如果普通人是1(恐懼接受度)，那兌換能力後變成5，這樣是否可行呢？但若激發潛能，恐懼接受度就會提升，畢竟解開了基因鎖(或激發潛能)，所以就可以接受更高的恐懼，這樣一來，也不需要兌換能力了啊！

但是如果沒有各種能力的出現，故事也就失去一大特點，無限恐怖中最讓人著迷的就是各種能力的搭配，所以有各種能力這點一定要保留。

所以換個方式，改成巫師空間，巫師是用智慧與知識走在進化的道路上，並不是說就忽視肉體的力量，像練體巫師或者是血脈巫師也是走在肉體的進化到路上，空有肉體沒有智慧和知識是不行的，所以改成巫師空間。

再者，作者認為既然要進化，這些養殖小隊、惡魔小隊、天使小隊，甚至所有小隊都不應該存在，巫師是一個獨立的個體，是一人走在進化的道路上，合作只是為了更好達成目標，所以不能以團隊的方式去建立每個人的能力，再者，養殖小隊之所以能成立，也只是因為前者比後者更先來到這個世界，利用優先權的方式，一種非自身努力的結果來成就自己，這絕對不是一個想要人類進化該有的方式。

故事：一位穿越成為巫師，並且成就無上偉業後回到地球，他製作了一件神器"巫師空間"，希望能幫助地球走上進化之路。

愛，是一個故事(Love Is A Story)
              Robert J. Sternberg, Ph.D.

Date: 20160316
Version: 1

一本介紹愛的關係的書籍

用故事來闡述愛的關係

這一本書是在成功嶺新訓的時候，講座推薦的一本書，起初我以為是一本介紹甚麼是愛的書，但其實不然。作者在一開始對愛的定義是由三種元素構成，親密、熱情、承諾，也就是這個讓我以為作者可以透過這三種元素來分析"愛"，但後來作者改變了想法，認為愛是一種關係，愛是一個故事，愛有各種故事，每一對之間都有屬於他們的愛的故事，因此，作者介紹了26種愛的故事，而每一種愛的故事都提供了兩則真實的故事讓讀者了解，並且作了量表來讓讀者可以測驗一下自己是屬於哪一種愛的故事，人是盲目的，很難自己了解自己是處於哪一種關係中，因此才需要量表的幫助，但作者也說了這26種只是其中一部份，世界上有許多各式各樣的愛的故事，這需要人們自己的體會。

說真的，看完這本書，我還是不了解甚麼是"愛"，因為學校從來沒教過甚麼是愛情，沒教過如何教男女朋友，這也是我一直無法理解的東西，到底是甚麼樣的因素讓兩個毫無相干的人變成男女朋友，為什麼這兩個毫無相關的人會彼此信任呢？為什麼有人認識沒多久就結婚了呢？怎麼可能在這麼短的時間內了解一個人呢？正所謂知人知面不知心，你怎麼了解一個人的內心在想甚麼呢？你怎麼可能去相信一個你不了解的人？你怎麼會知道哪天你在睡覺時，你的枕邊人從枕頭底下抽出一把刀，面無表情地往你身上捅下去(BTW，這是我的夢)，你有想過會發生這樣的事嗎？這也是我一直無法理解的事情。希望哪一天我可以了解到甚麼是"愛情"。

新訓+專訓心得

Date: 20160315
Version: 1

這一篇主要是講述對於新訓和專訓的心得，上兩篇比較像是對於新訓和專訓的一些描述。

我一月底畢業，二月過完年就要入伍了，在入伍的前一個禮拜，我常常腹瀉而且還有耳鳴的現象，我還以為我都不緊張呢！沒想到我會緊張成這樣，不過，幸運的是我有高中同學要一起入伍。
入伍之後，對於大部分的事情我都還蠻好適應的，唯一沒辦法的是睡覺，我只要有噪音就會沒辦法入睡，因此，入伍的前四天根本都沒睡，大概從第五天開始才慢慢地睡了一點，也多虧有成功嶺的這段經歷，出來成功嶺之後，不管是專訓還是在替代役中心，我都睡得還不錯，這真的要感謝成功嶺的這段經歷。
我的中隊是二中隊，不管是區隊長還是分隊長其實人都還不錯，如果你有問題都可以詢問他們，他們也會盡可能的幫助你，所以我其實對成功嶺這段經歷是不錯的，沒有奇怪的隊長，也沒有奇怪的同梯，大家都很好相處。
在成功嶺期間，曾因為生病要去轉診，這經驗是蠻難得的，當你坐在遊覽車上駛出成功嶺之後，看到路上各式各樣的商店，你會發現原來我還在世上，並不是與世隔絕的，那種感覺真的是無法形容出來的，雖然生病不太好，但這樣的經驗也是頗難得的。
在放假前，看著其他人先行離開，那種感覺真的不是很爽，還好我是因為要等我同學才一起留下來，所以我倒是覺得也還好，當然，如果可以早點走當然是早點走啊！
在成長體驗營中，最讓我難忘的是許願池和垂直降落，站在高處且身上只有一條繩子綁住時，要往前奮力一跳抓住欄杆，那真的是非常刺激，而垂直降落時，要背部朝後，往後仰的慢慢走下去，那時後說真的非常緊張，怕手一滑繩子鬆了，我就直接掉下去了。
而專訓的時候，我只能說非常的爽啊！從一個有各種規定的地方，到一個沒人管你的地方，那真的不是普通的自由啊！除了上課之外，其他時間沒有人管，你愛去哪就去哪？真的比其他替代役專訓爽上好多呢！
整體來說，我認為成功嶺要給我們的是一種紀律，當我們還沒進去前各種散漫，但當我們去來之後，才了解到自由是多麼難能可貴的。

小說背景設定-2

Date: 20160326
Version: 1

主題：天使與惡魔

虛幻世界(天堂與地獄) vs. 物質世界(人界)

虛幻世界依靠物質世界而存活

第N次聖戰(宗教 or 種族)

天使/惡魔 => 憑依 人類
                        附生
                        復生
                        寄生
                        共生
                        融合

但在人類世界不會有善惡大戰、種族對立

天使/惡魔
1. 從小到大，與人類有高度契合
2. 成熟體，與人類協同合作

能力：與人類共同產生(參考"武器種族傳說"、"驅魔少年")，當戰死時，天使/惡魔脫離，能力保留，尋找下一位適合者，能力可能與下一位適合者產生變化

天使種族與成長
惡魔種族與成長

第三類：天使、惡魔、魔鬼

替代役專訓

Date: 20160312
Version: 1

我選擇的役別是農業服務役，需用機關為農委會動植物防檢局，服勤單位是桃園市政府，專訓是一個禮拜，因為我們的服勤單位不在台北，所以非台北地區服役的替代役是住YMCA台北青年國際旅館。

專訓期間大概是這樣的，星期日下午回成功嶺，但不包晚餐和盥洗，所以請先吃飽洗完澡再回去，隔天一大早會要我們把寢具整理出來要送洗，然後發一些麵包當早餐之後，就背著黑大去集合，流程跟役別甄選一樣，喊到你的役別就出去集合，之後會由需用機關的人員帶你們到要專訓的地點，之後專訓就是在上課，早上一節，下午兩節，上一些關於之後服勤的一些業務資訊，最後星期五早上會進行考試，也就是專訓成績，但因為我們已經分好的原故，所以考試只是一種形式罷了，下午則是由服勤單位派人來接送。所以嚴格說起來，專訓只有三天，加上第一天的撥交，最後一天的考試和撥交，總共五天。

我們的服勤單位是在市政府農業局農務科，撥交之後會帶到桃園體育館居住，也就是之後一年的住宿地點，環境跟大學宿舍類似，4~6人一寢，床組和櫃子都有點舊了，沒有專屬的桌椅，而且需要路由器(因為只有一條網路線)，至於住宿管理辦法，因為我還沒入住的關係，等我住宿後我在進行補充管理辦法和住宿相關事宜。

替代役新訓

Date: 20160305
Version: 3

新訓16天終於結束了，也順利回到家了，趁六日有空把新訓的相關事宜紀錄一下。

我是161梯一般替代役，進入台中成功嶺受訓16天。

我會以條列式的方式進行整理，有比較重要的會再補充。

1. 第一天主要是搭車、體檢、領用服裝以及分組(分成幾個分隊，例如: 我是第九分隊)，當天行程很趕，有非常多的事情要做，最後進入宿舍前會進行安全檢查，所以還是盡量不要帶入違禁品(雨傘也是)，其中，領用服裝都是領前人留下來的衣服，在裡面內衣、內褲和襪子為基本款，每天送洗，但其他的部分大概是五六天洗一次，包括運動服和制服，所以進行要先習慣不怕髒。

2. 從第二天開始，幹部們都會開始要求各種紀律來把我們在外面的懶散驅除，所以在裡面就是聽話，一個口令一個動作，不要多作也不要少作，這樣會過得比較輕鬆。

3. 從第二天開始，會開始上一些課程與基本教練(立正稍息之類的)，日常生活大致上如下：起床(6:00 a.m.)-->跑三千-->吃早餐-->上課-->吃午餐-->午睡-->上課-->吃晚餐-->補教時間(較為輕鬆)-->盥洗打電話-->就寢(9:40 p.m.)-->循環，生活很規律的。

4. 裡面的飯菜，基本上沒有甚麼味道，但為了補充體力，能吃多少是多少，最主要是為了可以正常大號比較重要。

5. 之後會教替代役之歌，然後會進行替歌比賽，有得名有加分，只取六名，盡量就好。

6. 另外補充，裡面會有器材班和打飯班，器材班主要是班一些上課用的器具為主，而打飯班則是負責早午晚三餐進行打飯與飯後收拾為主，最後是過水班，主要是用各分隊輪流的方式來負責飯後洗碗筷的，本人是器材班，說真的器材班還滿爽的，平常就搬搬東西，還不用過水，只是我這梯內務被扣暴，所以大部分都是正常假和罰二，有點慘，而打飯班真的很辛苦，午睡又會被壓縮，真的很累，但是有加分。

7. 說到加分，就要講到正常假是4點離開成功嶺6點回來，罰二是6點離開6點回來，榮二是2點離開6點回來，榮四是12點離開6點回來，榮六是12點離開8點回來，罰四是6點離開4點回來，剛進去的時候，就會有人說才差兩個小時有差嗎？你待完16天你就會發現真的有差，看別人先離開心裡真的很幹！！！

8. 三千越野徒手跑步，如果平常沒在運動，一下子跑上下坡，很容易就受傷了，在加上這次A型流感很嚴重，每天醫護所滿滿的都是人，嚴重的或裡面不看的就會要你去轉診，會到附近的國軍醫院進行看診，大概半天就會不見了。

9. 此外，還有戰鬥阿嬤的課，誠心建議真的要去參加一下，我們最後一次的時候，剛好她的女兒沒辦法上課，我們就跟老師聊了一整節課，超有趣的。

10. 最後，當然是大家最關心的役別甄選，役別甄選主要流程如下：集合-->按照想要選擇的役別分別出去(前方會有人拿告示牌，跟著走就行了)-->進行排序(如果超過就進行抽籤)-->第一階段時間結束-->集合-->第二階段開始-->流程同上-->第二階段結束-->若還是沒有選到，隔幾天後，會統一進行抽籤分發。役別甄選的公告在頭幾天就會發下來了，趕快確認一下自己想選的役別，之後上課會統一公告這梯次碩博學士的人數統計，例如：161梯的博士畢業18、博是肄業14、碩士畢業277、碩士肄業254、學士畢業大概800左右、學士肄業大概500左右，然後你就可以自己算算看，然後到了現場之後問問其他鄰員的學歷，看看自己有沒有機會上。以161梯為例，這次需用人數比役男多50名，所以理論上都會上，而且這梯是今年第一梯再加上有的機關是一年一梯，所以有很多新的需用機關可以選，而且選完後之後的學弟們就選不到了，所以第一梯進去也是有好處的，選完後會告之專訓地點與時間，之後會於撥交日統一帶出，另外，有的役別連地點都會確定好(例如：農業試驗所就是在台中霧峰，之後也不用選地點，像消防役或教育役就是之後還要分發選地點)，那專訓就爽爽過就可以了，如果是之後專訓才要分發的話，你學顆測驗的成績包含軍事訓練成績和專業訓練成績都要考好，因為是按照分數高低選擇役別，分數高的先選這樣。

11. 再來是考試了，總共四個項目，三千平地徒手跑步(跑操場，不用喊口號)、基本教練(立正、稍息、右轉、左轉、後轉)、學科測驗、日常內務評比，3000平地20分鐘內很簡單，基本教練平時上課練好就行了，學科測驗看考古題就可以了，日常內務評比只有10%其實還好，只會影響到假別而已。

12. 在軍中唯一有的甜食，就是喉糖，唯一像糖果的東西，除此之外，大概還有三個途徑可以吃到，第一個是肝膽相照，可以去買零食來吃(我們分隊因為太吵結果取消了)，再來是入營的當月壽星有生日蛋糕可以吃，最後是去捐血，有飲料和餅乾可以吃。

13. 此外，還有所謂的成長體驗營，不外乎是一些攀岩、許願池、高空垂降之類的，行程是一天，如果是幹訓班的話，會有三天，不巧的是當天早上是3000公尺測驗，理論上有成長體驗營的話，當天是不會有跑3000的活動，只是剛好排到要考試，所以也沒辦法，另外，成長體驗營是在戶外，太陽很大，請記得隨時補充水分避免中暑。

14. 最後列一下比較重要的東西，正常應該知道的就不列了，1)喉糖(超重要，有事吃喉糖，沒事吃喉糖，買多一點吧)、2)有燈光的手錶(應該你會很早起來摺棉被，很暗想看時間就需要有燈光才行)、3)考古題(上課沒事可以看考古題，我看了所以我滿分)、4)A4格式的漫畫小說紙上益智遊戲(上課真的很無聊，沒事可以玩玩)、5)牙線(有的隊可以帶，有的不行)、6)電話卡/手機/行動電源/隨身碼(打電話用的)7)衛生紙(多帶點，因為你可能會感冒，我帶了兩包，三包比較保險)。

15. 裡面打電話有兩種，用電話卡打公共電話，或者是打手機，打手機的話要站在廣場打，有隊長負責監看，你可能需要行動電源，電話卡可能打個幾次就沒了，如果需要可能要準備多張一點，最後，我沒有用手機也不是電話卡，我是用隨身碼，價格比電話卡和手機便宜，下個月再繳錢就行了，很方便，有興趣的可以去中華電信辦理，只是因為隨身碼很冷門，櫃台小姐也可能不是很懂就是了。

白色的力量3：柯P模式
                            柯文哲

Date: 20160214
Version: 1

柯文哲的第三本自傳

描述整個選舉過程中，團隊的運行方式。

這一本自傳主要是在講述在整個選舉過程中，柯文哲是如何帶領他的選舉團隊、制定選舉策略，以及SOP的重要性。這三本自傳所帶來的意義不同，第一本是訴說著他為什麼決定要參加選舉，而第二本則主要是在講述他的個人政見，最後第三本則是要表現他所代表的價值觀，讓其他人更能深入地了解柯P在想什麼，以及想要什麼。
這本書中講了許多語錄，我特地針對兩點作說明，「當你思考的不是個人利益，而是眾人的利益時，就會開始信仰SOP。」和「你把部下當賊看，你就真的變賊。」。

「當你思考的不是個人利益，而是眾人的利益時，就會開始信仰SOP。」

最近特別對這句有感觸，什麼是個人利益，就是你本身的獨特性，假如只有你會這項技能，你就具有獨特性，無法被取代，保有自己的價值在，這樣的作法無可厚非，但這不利於後來者的學習與組織的發展，人人都想有保留自己的獨特性，避免被洪流淹沒，就像師傅怕徒弟把獨門技巧學走後另起爐灶一樣，怕失去自己的獨特性，所以只有在你會考慮到眾人的利益(自己以外)時，你才會認真的看待SOP，否則SOP就是一種剝奪自身價值的手段，這也讓我不禁想到《巫師之旅》中，巫師之所以能不斷蓬勃發展，跟他們本身對於知識的推廣與傳承不無相關，也因此我才會決定以這個目標看齊，試著把知識傳承下去，我以前也是那種會藏一手的以保持自身的獨特性，從畢業時實驗室交接工作可以看到，當你把自身辛苦的結晶交給下一個人時的排斥感，以及為了遵守諾言傳承知識的矛盾感，當你要接給下一個人的時後發現，對方怎麼這麼笨，但又因為要傳承知識而不得不靜下心來慢慢教導，這其中心靈的轉換真讓人五味雜陳啊！~~

「你把部下當賊看，你就真的變賊。」

而這句話令我想到實驗室的老闆，老闆他怕學生把實驗資料刪除，特地花錢買NAS，並叫我們把資料上傳到NAS中，從這裡可以知道他為什麼怕學生把資料刪除，因為他知道學生對他很不滿，怕學生銃康他把資料刪除，所以他才會先想到要把資料先存起來，以免之後被刪除。

Title

Identification of 2-oxohistidine interacting proteins using E. coli proteome chips.

Date: 20160206

Version: 1

Running Title

Identification of 2-oxohistidine interacting proteins

Abbreviations

The abbreviations used are: PTM, post-translational modification, MCO, metal-catalyzed oxidation, RAGE, receptors for advanced glycation end-products, Aβ, amyloid beta, AD, Alzheimer’s disease, GO, Gene Ontology, KEGG, Kyoto Encyclopedia of Genes and Genomes, BSA, bovine serum albumin, TBST, tris-buffered saline with tween 20, K_d, dissociation constant, AG peptide, AGAQVAHGNEVAG, SE peptide, SEAGVNHGSAGQA, IA peptide, IAVENVHAQGLA, Oxo-AG peptide, 2-oxohistidine residue in AG peptide, Oxo-SE peptide, 2-oxohistidine residue in SE peptide, Oxo-IA peptide, 2-oxohistidine residue in IA peptide.

Summary

Cellular proteins are constantly damaged by reactive oxygen species generated by cellular respiration. Due to its metal-chelating property, histidine residues are easily oxidized in the presence of Cu/Fe ions and H₂O₂ via metal-catalyzed oxidation, usually converted to 2-oxohistidine. We hypothesize that cells may have evolved antioxidant defenses against the generation of 2-oxohistidine residues on proteins, and therefore there would be cellular proteins which specifically interact with this oxidized side chain. Using two chemically synthesized peptide probes containing 2-oxohistidine, high-throughput interactome screening was conducted using the E. coli K12 proteome microarray containing >4200 proteins. Ten interacting proteins were successfully validated using fluorescence polarization assay through a third peptide probe of different sequence, as well as binding constant measurements. We discovered 9 out of 10 identified proteins seem to be involved in redox-related cellular functions. We also built the functional interaction network to reveal their interacting proteins. The network showed our interacting proteins were enriched in oxido-reduction process, ion binding, and carbon metabolism. A consensus motif was identified among these 10 bacterial interacting proteins based on bioinformatic analysis, which also appeared to be present on human S100A1 protein. The preferential binding of S100A1 with 2-oxohistidine over histidine was successfully validated using all three peptide probes, suggesting that the capacity to recognize 2-oxohistidine modification may be evolutionarily conserved from bacteria to humans. Besides, we found our consensus motif among our identified proteins, including bacteria and human, were all alpha-helix form and faced the outside of proteins which mean the motif has a chance to interact with the other proteins. The combination of chemically engineered peptide probes with proteome microarrays proves to be an efficient discovery platform for protein interactomes of unusual post-translational modifications, sensitive enough to detect even the insertion of a single oxygen atom in this case.

Introduction

The complexity of the proteome arises in a large part due to the hundreds of post-translational modifications (PTMs) already discover. Many PTMs are enzyme-catalyzed, such as phosphorylation, glycosylation, or ubiquitination (1, 2), but there are also numerous non-enzymatic PTMs caused by chemical reactions between reactive molecules and protein side chains, such as glycation, nitrosylation, and oxidation by reactive oxygen species (ROS) (3, 4). As protein side chains are enzymatically modified, there are generally specialized factors in the cell to recognize such changes. For instance, 14-3-3 family protein can recognize protein phosphorylation motifs (5) and various lectins can recognize protein glycosylation (6). However, recognition factors may also exist for non-enzymatic PTMs, such as receptor for advanced glycation end-products (RAGE) (7). In this study we seek to uncover cellular binding factors for 2-oxohistidine, the oxidized product of histidine, which is an important but less understanding non-enzymatic PTM.

The generation of ROS is an unavoidable consequence of cellular respiration, which leads to the oxidation of proteins, lipids, and nucleic acids (4, 8). ROS play regulatory roles in cellular signaling pathways under low levels (9), but high levels of ROS are cytotoxic and lead to the accumulation of damaged cellular components (10, 11). The reactions of proteins with ROS may lead to almost 100 side chain modifications (12, 13). Histidine is highly susceptible to ROS damage, because it has strong metal chelation affinities and often constitutes the binding site for metal ions (14, 15). The presence of H₂O₂ and redox-active metals (Cu and Fe) can lead to metal-catalyzed oxidation (MCO, also called Fenton-type chemistry), which converts histidine side chains to 2-oxohistidine (16, 17).

The conversion of histidine to 2-oxohistidine alters its charge state, hydrogen bonding property, and metal chelation affinity, and hence may have seriously impact on protein structure and function. The net reaction is oxygen insertion (+16 Da), which makes it an irreversible PTM. It is unclear if cells simply tolerate such damages on histidines or employ active mechanisms to recognize them and use them as redox sensors or as damage markers for promoting protein degradation. The only known biological function of 2-oxohistidine is to serve as a redox sensor on bacterial transcription factor PerR (18), while other studies have used 2-oxohistidine as a stable marker of protein damage during oxidative stress (12, 19).

Judging by the potential biological significance of 2-oxohistidine modification, we hypothesized that there may be cellular factors to recognize it. Previous research on 2-oxohistidine had been impeded by the difficulty in generating this side chain with reasonable yields. Recently, we managed to greatly improve the yield of 2-oxohistidine conversion by optimizing MCO reaction conditions using the copper/ascorbate system (20), allowing us to synthesize and purify peptide probes containing 100% 2-oxohistidine for this study.

Here, we used 2-oxohistidine-containing peptides to mimic the oxidative conversion of histidine residues on native proteins. Then, we utilized the E. coli K12 proteome chip to identify 2-oxohistidine-interacting proteins via high-throughput screening, and the interactors turned out to be largely involved redox-related metabolism. From the bacterial interactors we predicted a consensus binding motif, which could be validated across different species and correctly predicted S100A1 as a human binding factor for 2-oxohistidine. Thus, recognition of 2-oxohistidine appears to be an evolutionarily conserved capacity from bacteria to human.

Experimental Procedures

Fabrication of E. coli K12 proteome chip

The high throughput protein expression, protein purification, and protein printing were modified from the previous study (21). Briefly, we expressed and purified E.coli K12 protein in 96-well plate format and subsequently printed the proteome microarray. All purified proteins were spotted in duplicate on each aldehyde slide (BaiO, China) by SmartArrayer 136 (CapitalBio, China) at 4°C. After printing proteins, the proteome microarray chips were kept at 4°C for protein immobilization on the slides for 12 h. In the end, the chips were stored at -80°C before probing with samples.

Peptide oxidation

Solutions containing 1 mM peptide, 5 mM Cu2+ and 200 mM sodium ascorbate were exposed to air with gentle shaking at 37 °C for 24 hrs (AG and SE peptide) or 6 hrs (IA peptide). The oxidation reaction was quenched with 20 mM EDTA and analyzed by reverse-phase HPLC (10-30% acetonitrile and 0.1% TFA in water, C18 column from Dr. Maisch, Ammerbuch, Germany) to determine the reaction yield. For LC-MS/MS analysis of crude reaction mixtures and HPLC fractions, 10 μL samples was acidified with 2 μL 10% TFA and desalted with ZipTip (Millipore, Billerica, MA) following manufacturer’s protocols. Oxidized peptides were purified by semi-preparative HPLC (C18 column, Dr. Maisch). LC-MS/MS experiments were conducted under previously reported conditions (20).

Peptide labeling

        Oxidized and non-oxidized peptides were dissolved in 50 mM sodium borate buffer at pH 7.5 and analyzed by HPLC to determine peptide concentration by 210 nm absorbance. DyLight-conjugated NHS esters were dissolved in anhydrous DMF to 10 mg/mL and added to peptide solutions for 1 hr incubation at room temperature, at the following fluorophore/peptide ratios: DyLight 650:AG =3:1, DyLight 650:SE = 5:1, DyLight 650:oxo-IA = 1.5:1; DyLight 550:oxo-AG = 5:1, DyLight 550:oxo-SE = 7:1, DyLight 550:IA = 3:1. Labeled peptides were analyzed and purified by HPLC as described above. Labeled products were verified by LC-MS/MS, and quantified by absorbance measurements based on fluorophore properties.

E. coli K12 proteome chip assays with 2-oxohistidine peptides

The chips were first blocked with 3% bovine serum albumin (BSA) (Sigma-Aldrich, US) for 5 min. Ten μM of DyLight^TM 550-conjugated 2-oxohistidine peptide and DyLight^TM 650-conjugated non-oxohistidine peptide were probed together onto the chip with LifterSlips^TM (Thermo Scientific, US) at room temperature for 45 min. Finally, the chips were washed by Tris-buffered saline-Tween 20 (TBST) in an orbital shaker three times and 5 min each time. The chip was dried by centrifugation and then scanned with a LuxScan^TM microarray scanner (CapitalBio, China). Signal intensities, foreground median subtract background median, were acquired and analyzed using GenePix Pro 6.0 software. Then, we used quantile normalization to normalize the signal intensity from both 2-oxohistidine containing probes and non-oxohistidine containing probes. To identify positive 2-oxohistidine interacting proteins, four cutoffs were set. 1) The signal from experimental groups was greater than 1.5 standard deviations away from the mean for experimental groups. 2) To get the large signal difference between experimental groups and negative controls, the delta, defined as signal difference between experimental group and control group, was greater than 1.5 standard deviations away from the mean for all deltas. 3) To exclude the non-specific binding to 2-oxohistidine residue, the signal from the negative control was less than 1.5 standard deviations away from the mean for control group. 4) To remove the irreproducible hits among triplicate chip assays, the student’s t-test p-values between experimental groups and negative controls were less 0.05.

Heat Map

The R programming language (22) was used to display heat map. The data was presented by signal intensity of foreground subtract background. The gplots package (23) was used for classifying 2-oxohistidine containing peptides and non-oxohistidine containing peptides in hierarchy.

Functional interaction analysis

The identified proteins were used for functional interaction analyses by using EcID (24) and Cytoscape (25). Briefly, the files of EcID entities and EcID pairs were downloaded from EcID database. Before mapping identified proteins to their EcID entities and EcID pairs, we removed the pairs which based on the prediction mode, such as phylogenetic profiles, gene neighborhood, mirror tree, insilicon 2 hybrid, or context mirror. After mapping, we used Cytoscape to generate the functional interaction network, and visualized the identified proteins and their interacting proteins. Later on, we used AmiGO 2 (26) and KOBAS 2.0 (27) to generated gene ontology (GO) (28) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (29) results, respectively.

Fluorescence polarization assay

After blocking the 96-well black plate (Thermo Scientific, US) with 1% BSA at room temperature for 1 h, the identified proteins was added to the plate. The concentrations of 10 identified proteins (ThrS, YqjG, YajL, HemE, IlvA, PrpD, Zwf, Eda, Gor, and PqqL) were 12.0, 25.7, 10.7, 15.6, 3.4, 18.6, 19.5, 11.8, 26.1, and 5.9 μM, respectively. And the concentrations of BSA, as a negative control, were as same as the protein they compared to. Ten nM of DyLight^TM 550-conjugated 2-oxohistidine peptide was incubated with protein or BSA in a Micromixer MX4 (FINEPCR, South Korea) at room temperature for 1 h. After incubation, the degree of polarization of each well was detected by a Synergy 2 (BioTek, US), using an excitation wavelength of 540 nm and an emission wavelength of 590 nm with a dichroic mirror of 570 nm.

Measurement of dissociation constant (K_d)

Identified proteins and S100A1 (Abnova, Taiwan) were printed on aldehyde chips in a multiple-well format. After printing, the chips were immobilized at 4 °C for 12 h and then stored at -80 °C. The printed chips were blocked at room temperature for 5 min with 3% BSA. Two folds serial-diluted DyLight^TM 550-conjugated 2-oxohistidine peptides, DyLight^TM 650-conjugated non-oxohistidine peptides, and quenched fluorescent dyes were probed onto the wells of the chip individually with Multi-Well Microarray Hybridization Cassettes (Arrayit, US), and incubated at room temperature for 45 min. The fluorescent dyes, DyLight^TM 550 and DyLight^TM 650, were already quenched by 5M Tris-HCl (Bionovas, Canada). To check whether calcium affects interaction between S100A1 and 2-oxohistidine, 1 mM CaCl₂ was added in the assay buffer. After washes with TBST, the chips were dried by centrifugation and then scanned with a microarray scanner. The K_d value was calculated by double-reciprocal plot analysis which y is one divided by fluorescence intensity, and x is one divided by peptide concentration. Set the regression line formula in the form of y = ax, which “a” is the slope of regression line. The K_d value will be “a” times concentration of identified protein.

Motif Search with GLAM2

All identified proteins were converted to FASTA format and analyzed by Gapped Local Alignment of Motifs (GLAM2) (30) for surveying consensus motif. The parameters of GLAM2 were set as default. The resultant motif was then searched in entire E. coli K12 proteome and human proteome by GLAM2SCAN (30).

Protein 3D structure and secondary structure prediction

All protein 3D structures were provided by their provider (31-38) and RCSB PDB (39). The colors in protein 3D structures were visualized by RasMol software (40). We used the EcoGene 3.0 (41) which contains the QUARK prediction method (42) to predict the secondary structure of those proteins which do not have protein 3D structures.

Results

Many researches revealed that the 2-oxohistidine residue had been discovered in several peptides or proteins (16, 43-51). We used the E. coli K12 proteome chip to identify proteins which can bind specifically to 2-oxohistidine residue. To accomplish our purpose, we fabricated the E. coli K12 proteome chips, generated the 2-oxohistidine containing peptides, and probed these peptides with E. coli K12 proteome chips. After identified the positive hits, we used fluorescence polarization assays to validate the interactions and measured the binding affinity by dose-response measurements. Then, we surveyed the consensus motif among these identified proteins and applied to human proteome to look for the possible human 2-oxohistidine interacting proteins. Finally, we used the functional interaction network to find out the possible interacting proteins and used GO and KEGG to figure out possible process and pathway (Fig. 1).

Oxidation of peptide histidine residue

Histidine residues are placed in the middle of 12-mer or 13-mer peptides to eliminate possible charge effects at N-terminus and C-terminus, creating a context similar to proteins. Easily oxidized amino acids, such as methionine, cysteine, tyrosine, tryptophan phenylalanine, lysine, and arginine, are avoided. Three peptides containing a single histidine residue and random selections of other residues, namely AGAQVAHGNEVAG (AG), SEAGVNHGSAGQA (SE), and IAVENVHGGLA (IA), were used for chip assays. We carried out MCO reaction using the copper/ascorbate/air system shown in Figure 2. The HPLC yield of mono-histidine peptides AG and SE were around 10%, and for IA peptide around 20% (Fig. 2).

E. coli K12 proteome chip assays

To investigate 2-oxohistidine interacting proteins, AGAQVAH*GNEVAG (Oxo-AG peptide) and SEAGVNH*GSAGQA (Oxo-SE peptide) were conjugated to DyLight^TM 550 fluorophore molecular probes. Non-oxidized AG and SE peptides were conjugated to DyLight^TM 650 as negative controls. In the chip assay, 2-oxohistidine containing peptide and its negative control were probed with E. coli K12 proteome chip in triplicate (Fig. 3). The examples of 2-oxohistidine interacting proteins compared with non-oxohistidine containing peptide profiling were shown in Figure 4.

To identify the specific hits to 2-oxohistidine peptides, we set several cutoffs. First, we chose the hits had strong intensity in experimental groups. Second, we wanted the hits had high signal in experimental groups and low signal in negative controls. Thus, we chose the hits had large difference between experimental groups and negative controls. Third, although we chose the hits had large difference between two groups, there still were some strong signals in negative controls. To exclude this kind of non-specific binding to 2-oxohistidine residue, we removed the hits which greater than 1.5 standard deviationa away from the mean for negative controls. Fourth, in order to have reproducibility results among triplicate chip assays, we excluded the hits which had large variances as we described in the section of experimental procedures. Under such criteria, 38 and 20 protein hits were found to bind oxo-SE peptide and oxo-AG peptide, respectively (supplementary Table S1-S2). To avoid the non-specific binding due to the different peptide sequences, we chose the hits shared by both 2-oxohistidine peptides among those proteins. Only 10 proteins (ThrS, YqjG, YajL, HemE, IlvA, PrpD, Zwf, Eda, Gor, and PqqL) were identified by both 2-oxohistidine containing peptides (Table 1).

We used heat map to visualize the intensity of these 10 identified proteins among 2-oxohistidine and non-oxohistidine containing probing results (Fig. 5). The heat map shows that our 10 identified proteins clearly classified the 2-oxohistidine peptides from non-oxohistidine peptides.

Functional interaction analysis

We exploited EcID to find our 2-oxohistidine interacting proteins’ partners that indirectly interacted to 2-oxohistidine. The EcID database (Escherichia coli Interaction Database) (24) provided a framework for the integration of several interactional source, including EcoCyc (metabolic pathways, protein complexes and regulatory information), KEGG (metabolic pathways), MINT and IntAct (protein interactions), high-throughput experiment (protein complexes), and iHOP (text mining).

In this study, we only selected interactions from experimental mode which was proved by many databases and the results would be more reliable and confident. We chose the interacting proteins that had at least interacted 3 out of the 10 identified 2-oxohistidine interacting proteins. As shown in Figure 6, four 2-oxohistidine interacting proteins (thrS, zwf, eda, and ilvA) were ‘‘hubs’’ that connected many interacting proteins in the network. From this functional interaction analysis, 26 interacting proteins were found to have interactions with at least 3 out of the 10 identified proteins. We further analyzed this functional interaction network, including 10 identified proteins and 26 interacting proteins, by using AmiGO 2 (26) and KOBAS 2.0 (27) to provide the GO (28) and KEGG (29) results, respectively (supplementary Table S3-S5). Interestingly, fifteen out of the 36 proteins (~40%) were in the oxidation-reduction process, which shows significant enrichment (p < 0.05). Table 2 summarizes the related GO terms and KEGG pathways. Oxidation-reduction process is a metabolic process that involved in the transfer of electrons between chemical species (52). This result suggested that our identified 2-oxohistidine interacting proteins and their interacting proteins from the network may involve in the oxidation-reduction process. In the molecular function, ion binding and cofactor binding were enriched in our network. This result suggested that our proteins may interact with metal ion which can lead to MCO reaction. Besides, oxoacid metabolic process and carbon metabolism were also discovered. These kinds of metabolism usually accompany with energy metabolism that the reducing power and ROS may also carry out in the process (53). Changes to the oxidation state of a molecule were frequently carried out as a secondary metabolite were synthesized or modified (54). Therefore, the biosynthesis of secondary metabolites was also enriched in our interaction network. These data showed that identified proteins and their binding proteins may involve in the redox process or the oxygen sensitive environment to responsible for such kinds of oxidation change or be a protector or sensor to the oxidative stress.

Fluorescence polarization assays

Although there were positive results in the chip assays, we still could not exclude the possible bias of this kind of heterogeneous approach. Fluorescence polarization assay is a kind of homogeneous binding detection methods to mimic the interaction between two compounds in the cellular environment (55-59). Fluorescence polarization assays, investigation of the binding between two molecules were used to validate the 10 identified proteins in this study. Once the protein bound to fluorescent 2-oxohistidine containing peptides, a high degree of polarization was expected. As shown in Figure 7, all the 10 identified proteins had higher polarization than the negative control, BSA. Besides, the polarization distribution of two oxidative peptides was similar to each other. It indicated that interaction between proteins and 2-oxohistidine was not affected by different peptide sequences. The result confirmed that 10 identified proteins can bind to 2-oxohistidine in both AG and SE peptides.

Measurement of binding affinity

Dissociation constant (K_d) described the propensity of a ligand-protein complex to dissociate reversibly into its components. We measured the K_d of these identified proteins to oxidative peptides, normal peptides, and quenched fluorescent dyes by dose-response measurements. Fluorescent 2-oxohistidine containing peptides with different concentrations probed onto the slide, where the identified proteins were immobilized (supplementary Fig. S1A). Using double-reciprocal plot analysis, we calculated the K_d values for all identified proteins (supplementary Fig. S1B). The same procedures were done in normal peptides and fluorescent dyes, too. The result showed our 10 identified proteins had a strong affinity to 2-oxohistidine from 10^-8 to 10^-10 M, especially the hemE protein which had the highest K_d (~10^-10 M) in both 2-oxohistidine containing peptides (Table 3). We also found our proteins slightly preferred oxo-SE peptide than oxo-AG peptide, but the difference of K_d was not greater than one order of magnitude. On top of that, the K_d values from oxidative peptides were significant difference to the normal peptides, and quenched fluorescent dyes (p < 0.05). To check the interaction between 2-oxohistidine and identified proteins again in order to be certain. We used a third peptide, IAVENVH*QGLA (Oxo-IA peptide) and its negative control (IAVENVHQGLA, IA peptide), which had different peptide sequence and we also swapped their fluorescent dyes to each other to avoid the influence of fluorescent dyes. The result also showed the statistically significant difference to its negative controls (p < 0.05). This indicated that our 10 identified proteins had a strong binding affinity to 2-oxohistidine, and were not affected by different peptide sequences and different fluorescent dyes.

Motif Searching in E. coli proteome and human proteome

Based on fluorescence polarization and binding affinity results, we performed the GLAM2 (Gapped Local Alignment of Motifs) (30) to survey whether a consensus motif among these identified proteins. In this study, we found the consensus motif among these identified proteins is [SD][QV][AEDT]A[YIL][CE][AK][ARL][MV][AHK]?[KET][LV] [AYLF]E (Fig. 8). In addition, we used this motif to query entire E. coli K12 proteome by GLAM2SCAN (30). The result showed top ten ranking proteins containing this motif were identical to our identified proteins (Table 4). This indicates that motif was significantly unique in the entire E. coli K12 proteome (p < 0.05). We also applied this motif to entire human proteome, and found the ranked top one protein is S100 Calcium Binding Protein A1 (S100A1), which is a member of the S100 family (supplementary Table S6).

After motif screening in E. coli and human proteome, we further investigated the secondary structure of the motif in our identified proteins and S100A1 by using protein 3D structures (Fig. 9). However, there were 3 proteins (hemE, zwf, and pqqL) were not available. For these three proteins, we used the QUARK prediction method to predict their secondary structures. By proteins 3D structure analysis or QUARK prediction, the result showed that this motif was usually an alpha-helix in these proteins except for yajL, which contains 36% beta-sheet and 64% alpha-helix in the motif (Table 5). Besides, we found these kinds of alpha-helix formed motifs generally faced the outside of the proteins which mean they had chance to interact with outside molecules. Our finding suggested that 2-oxohistidine recognized motif was an alpha-helical structure and conversed between E. coli and human.

K_d measurement between human S100A1 protein and the oxidative peptides

To validate the interaction of human S100A1 protein we found by GLAM2SCAN on entire human proteome, we calculated the K_d values according to dose-response measurements for all oxidative peptides, including oxo-AG peptide, oxo-SE peptide and oxo-IA peptide. The result showed that S100A1 protein had a strong affinity to all 2-oxohistidine containing peptides and significant difference to the other unoxidized peptides and fluorescent dyes (p < 0.05) (Table 6). The binding affinity of S100A1 to 2-oxohistidine were 10-fold to 100-fold higher than the negative controls, indicating that S100A1 actually had an ability to bind to the 2-oxohistidine. Since we knew S100A1 is calcium binding protein, we wondered whether calcium would affect the interaction or not. The result showed calcium was not involved in the interaction of S100A1 to 2-oxohistidine peptides or the other groups (p > 0.1). This suggested the E. coli K12 proteome chip was able to be a feasible platform for motif screening in cross-species studies.

Discussion

Enzymatic and non-enzymatic PTMs are comparable in their diversity and chemical complexity, but past research efforts have mostly focused on the former, leaving a huge gap in our understanding of biological phenomena associated with non-enzymatic PTMs. Even though non-enzymatic PTMs are not generated by enzyme actions, there may still be specific enzymes to chemically reverse such modifications, or specific receptors to detect such modification. For example, the chemical oxidation of methionine to methionine sulfoxide can be reduced back to methionine by specific reductases MsrA and MsrB (60); RAGE can recognize protein glycation and lead to inflammatory responses (7). However, there are still many non-enzymatic PTMs for which the biological functions are little known.

Among non-enzymatic PTMs, 2-oxohistidine is particularly interesting because of its minimal size, involving the insertion of just one oxygen atom. It probably represents the smallest atom-scale alteration associated with a known PTM, and we investigated if cells have evolved the ability to monitor such a small change on the surface of proteins. Because histidine often plays critical roles in protein function, both structurally and catalytically, we hypothesized there would be cellular factors that specifically recognize 2-oxohistidine side chains, and this hypothesis was tested with specially synthesized peptide probes, and E.coli proteome chips.

Using three peptide probes with homogeneous 2-oxohistidine modification, we were able to identify 10 proteins that show preferential binding for 2-oxohistidine-containing peptides over non-oxidized control peptides (Table 1). Since these three probes have very different flanking sequences, it is very likely that we have identified proteins which specifically recognize side-chain differences between 2-oxohistidine and histidine, and we will refer to them as 2-oxohistidine recognition factors. Before this study, the recognition factors of 2-oxohistidine had never been proposed or identified.

In theory, the recognition of 2-oxohistidine could play several different biological roles. First, it may act as a redox sensor, similar to S-nitrosylation (61). Secondly, it may identify oxidatively damaged proteins and mark it for degradation. Third, it may trigger cellular stress responses and antioxidant pathways. Although there is no known involvement of 2-oxohistidine in different E. coli physiological pathways, several of the recognition factors in E. coli appear to be related to redox pathways and antioxidant pathways.

Among the 10 putative recognition factors identified via proteome array, 9 seem to be involved in redox-related cellular functions. Gor is a glutathione reductase, involved in the generation of glutathione, which maintains the reducing environment of the cell (62). YqjG is glutathionyl hydroquinone reductase, which utilizes glutathione to reduce a wide range of organic molecules (38). HemE is an uroporphyrinogen decarboxylase involved in the biothesis of the heme group, which is an important cofactor for antioxidant enzymes like catalase and peroxidase (63). Zwf is a glucose-6-phosphate dehydrogenase, which helps supply NADPH through the pentose phosphate pathway (64), and NADPH is a cofactor used as a reducing agent by many metabolic enzymes (65, 66). PqqL in E. coli is a putative zinc metalloprotease, but functionally it may be similar to pqqF in Klebsiella pneumoniae, which has a supportive role in pyrroloquinoline quinone biosynthesis (67). Pyroloquinoline quinone is a redox cofactor that provides reducing power for the cell, and also a ROS scavenger (68).

YajL is an anti-oxidative-stress chaperone, which promotes disulfide formation to help maintain order in the thiol proteome (69). Interestingly, the human homolog of yajL, DJ-1, is also an anti-oxidative stress protein, and its mutations are known to cause familial Parkinsonism (70). On the other hand, ilvA and thrS are both involved in threonine metabolism, and known to be regulated by oxygen levels in the cell. IlvA, a threonine dehydratase, converts threonine to 2-oxobutanoate, and its promoter is activated by oxygen (71). ThrS is a threonyl-tRNA synthetase, and potentially also an oxygen sensor in the cell through Cys182 oxidation (72). Eda, Entner-Doudoroff aldolase (also called KDPG aldolase), is involved in the Entner-Doudoroff pathway that generates pyruvate and NADPH by consuming glucose. Eda is a multi-functional aldolase which also catalyze the addition of pyruvate to electrophilic aldehydes to detoxify harmful byproducts generated by oxidative stress (73).

PrpD, a 2-methylcitrate dehydratase, does not appear to be directly involved in redox functions, but it converts propionyl-CoA into pyruvate through the methylcitrate cycle (74), and pyruvate can be utilized by the aforementioned eda to detoxify oxidized organic molecules with aldehydes. Therefore, all 10 putative recognition factors for 2-oxohistidine identified here appear to be involved in supplying reducing power to the cell or in oxygen-sensitive regulation of carbon metabolism. This strongly implies that recognition of 2-oxohistidine in E. coli may play certain roles in redox sensing and metabolic regulation, but further experiments are required to elucidate its actual function.

Using motif analysis by GLAM2 and GLAM2SCAN, we identified putative 2-oxohistidine binding motif from these 10 recognition factors, which turned out to be: [SD][QV][AEDT]A[YIL][CE][AK][ARL][MV][AHK]?[KET][LV][AYLF]E. We further validated this binding motif by searching for the highest-scoring match in the human proteome, which turned out to be DVDAVDKVMKELDE on S100A1 protein, and we verified that S100A1 indeed exhibited 2-oxohistidine binding affinity. S100A1 is a calcium binding protein highly expressed in the brain and heart, and its calcium binding affinity is greatly enhanced by the oxidative nitrosylation of Cys86 (75). It is believed to regulate calcium and nitric oxide signaling in neuronal cells, affecting neurotransmitter release as well as inflammation (76). Interestingly, since S100A1 is also secreted extracellularly (77), it may bind to oxidized amyloid beta (Aβ) with 2-oxohisitidine side chains, which are released from extracellular senile plaques which trap metals and generate ROS (45, 78, 79). Since Aβ is known to cause calcium misregulation (80), oxidative stress (81), and inflammatory response (82) in the brain, the interaction between S100A1 and oxidized Aβ through 2-oxohistidine recognition may play a role in Alzheimer’s disease (AD) pathogenesis, which warrants future investigation.

Our preliminary evidence suggests that both bacteria and humans have cellular factors which can recognize 2-oxohistidine side chains, and a conserved binding motif has been putatively identified. Through the course of evolution, the recognition of 2-oxohistidine may carry important cellular functions related to redox signaling. We have also shown that E. coli K12 proteome microarray is capable of being exploited as a motif library for screening small molecule binding, and that even a single-atom modification on the molecule may be recognized. We expect a wide application of this approach for studying the interaction of other post-translational modifications, such as phosphorylation, methylation, acetylation, amidation, thiolation, sulfation, nitrosylation, as well as many non-enzymatic PTMs. With regard to 2-oxohistidine, future work is required to elucidate how single-oxygen insertion can be recognized on the protein surface, and how recognizing this modification regulates biological functions.

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Figure Legends

Figure 1. Overall strategy for the identification of 2-oxohistidine interacting proteins using E. coli K12 proteome chip. We expressed and purified ~4,300 E. coli proteins in high-throughput to fabricate the E. coli K12 proteome chip. We used an improved condition to obtain 2-oxohistidine peptides in high purity. 2-Oxohistidine peptides were then probed to E. coli K12 proteome chip and identified the preferential binding proteins. We also built their functional interaction network to investigate their biology. Fluorescence polarization assays were used to validate the identified proteins. We conducted dose-response fluorescence assays to measure the K_d of these proteins. Furthermore, we used GLAM2 to search consensus motif among these identified proteins and also applied this motif to entire E. coli K12 proteome and human proteome by GLAM2SCAN.

Figure 2. Summary scheme for the synthesis of 2-oxohistidine-containing peptides. The process was synthesized by using metal-catalyzed oxidation, and the histidine side chain on peptides was converted to 2-oxohistidine.

Figure 3. Schematic of E. coli K12 proteome chip assays with 2-oxohistidine peptide probes. To detect the 2-oxohistidine interacting proteins, E. coli K12 proteome chips were probed with 2-oxohistidine-containing peptides and un-oxidized control peptides labeled with different fluorophores. Each protein was printed in duplicate on the chips.

Figure 4. Representative images of the E. coli K12 proteome chips probed with 2-oxohistidine containing peptide (Oxo-SE peptide) and non-oxohistidine containing peptide (SE peptide). The representative positive hits (yqjG and thrS) and non-specific binding protein (yeiG) on the chip were enlarged from sample images of oxo-SE peptide and SE peptide, respectively. The contrast and brightness of images had been equally adjusted using the same parameters.

Figure 5. The heat map of 10 identified proteins. The heat map showed the classification of 10 identified proteins in oxo-AG, oxo-SE, AG and SE chip assay probing result. Each peptide probes had triplicate results. The R programming language and gplots package were used to display heat map.

Figure 6. The functional interaction network of the 10 identified proteins and 26 interacting proteins. The interaction pairs for 10 identified proteins were downloaded from EcID database, and functional interaction network was visualized by Cytoscape. We only showed the interacting proteins that interact with at least 3 out of 10 identified proteins, and 26 interacting proteins were identified. Four out of 10 identified proteins, eda, ilvA, zwf, and thrS, had many interactions and considered to be hubs. Square shapes represented the 10 identified proteins, and round shapes represented the 26 interacting proteins. The node color showed the number of interactions, the red is greater than 10 interactions, the green is greater than 5 interactions, and the others are yellow which smaller than 5 interactions. The thicker edge lines symbolized that more databases showed the interaction between 2 proteins.

Figure 7. Validation of the interactions between 2-oxohistidine peptides and identified proteins using fluorescence polarization assays. In fluorescence polarization assays, the polarizations of the tested proteins were compared with same concentration of BSA, as a negative control. A. The fluorescence polarization assays for oxo-AG peptide and identified proteins. B. The fluorescence polarization assays for oxo-SE peptide and identified proteins. The black bar is identified proteins and the gray bar is BSA. The asterisks mean the polarizations of the identified proteins were significant difference to the BSA control (p < 0.05).

Figure 8. Consensus motif among the 10 validated proteins. A motif [SD][QV][AEDT]A [YIL][CE][AK][ARL][MV][AHK]?[KET][LV][AYLF]E was identified by GLAM2. The table showed the protein sequences of 10 validated proteins aligned with consensus motif.

Figure 9. Protein 3D structure of E. coli identified proteins and human S100A1. Only 7 E. coli identified proteins (thrS, yqjG, yajL, ilvA, prpD, eda, gor) and human S100A1 had protein 3D structures. The protein 3D structures were provided by their provider and RCSB PDB, and visualized by RasMol software. Beta-sheets are shown in yellow bands; alpha-helices are shown as pink bands and random coil as white lines. The blue bands are the consensus motif we found by GLAM2. Only yqjG, yajL, prpD, gor and S100A1 were provided by homodimer structure. The other is the monomer structure.

Tables

Table 1. 2-Oxohistidine interacting proteins identified by E. coli K12 proteome chips. There were 38 and 20 proteins are identified by oxo-AG peptide and oxo-SE peptide chip assays, respectively. To avoid the non-specific binding due to the different peptide sequences, we only chose the hits were shared by both 2-oxohistidine containing peptides (oxo-AG peptide and oxo-SE peptide).

Accession ID	Protein Symbol	Protein Name	Protein Function
EG11001	thrS	Threonyl-tRNA synthetase	An enzyme involved in protein synthesis which is regulated by aerobic and anaerobic metabolisms
EG12746	yqjG	Glutathionyl-hydroquinone reductase	Reduction of organic small molecules
EG13272	yajL	Anti-oxidative stress chaperone	A covalent chaperone for thiol-containing proteome, also promoting disulfide formation
EG11543	hemE	Uroporphyrinogen decarboxylase	Involved in the synthesis of heme group, which is a critical cofactor for antioxidant enzymes
EG10493	ilvA	Threonine dehydratase	A metabolic enzyme that converts threonine to 2-oxobutanoate, regulated by an oxygen-responsive promoter
EG13603	prpD	2-Methylcitrate dehydratase	A metabolic enzyme in the methylcitrate cycle that converts propionyl-CoA to pyruvate
EG11221	zwf	Glucose-6-phosphate dehydrogenase	A metabolic enzyme in the pentose-phosphate pathway that supplies reducing power to cells generating NADPH
EG10256	eda	KDPG aldolase	An enzyme in the Entner-Doudoroff pathway, also a multi-function aldolase to detoxify aldehydes generated by oxidative stress
EG10412	gor	Glutathione reductase	An enzyme that generates glutathione to maintain a reducing environment in the cell
EG11744	pqqL	Putative periplasmic M16 family zinc metalloendopeptidase	An enzyme involed in pyrroloquinoline quinone biosynthesis, which is a redox cofactor that supplies reducing power

Table 2. Summary for functional analysis of 36 proteins from functional interaction network. The 36 proteins, including 10 identified proteins and 26 interacting proteins, were used to do the functional analysis. The GO and KEGG results were generated by AmiGO 2 and KOBAS 2.0, respectively. We summarizedthe related GO terms and KEGG pathways in this table. The entirely detailed information of GO and KEGG results were shown on supplementary Table S3-S5.

GO Term (Biological process)	ID	Protein involved numbers	p-value
Oxoacid metabolic process	GO:0043436	21	5.29E-08
Oxidation-reduction process	GO:0043436	15	5.03E-03
GO Term (Molecular function)	ID	Protein involved numbers	p-value
Ion binding	GO:0043167	27	3.43E-05
Cofactor binding	GO:0048037	15	2.38E-06
KEGG	ID	Protein involved numbers	p-value
Carbon metabolism	eco01200	11	2.89E-03
Biosynthesis of secondary metabolites	eco01110	19	1.25E-02

Table 3. K_d values for 2-oxohistidine peptides binding to identified proteins. All K_d values were determined by dose-response measurements. Different concentration of fluorescent oxidative peptides, normal peptides, and fluorescent dye were probed onto the chip which 10 identified proteins immobilized. Based on the dose-response, we could use double-reciprocal plot to calculate the K_d values. We also used the oxo-IA peptide, which was different peptide sequence and labeled different fluorescent dye, and its negative control (IA peptide) to confirm the binding between identified proteins and 2-oxohistidine.

	Oxidative Peptides			Normal Peptides			Fluorescent Dyes
Name	DyLight 550 oxo-AG	DyLight 550 oxo-SE	DyLight 650 oxo-IA	DyLight 650 AG	DyLight 650 SE	DyLight 550 IA	DyLightTM 550	DyLightTM 650
thrS	1.2E-8 ± 9.6E-10a	6.7E-9 ± 1.9E-9a	3.9E-8 ± 4.7E-9a	1.4E-7 ± 3.5E-8	2.5E-7 ± 8.3E-8	1.0E-7 ± 6.8E-8	8.5E-8 ± 1.1E-8	1.1E-7 ± 1.6E-8
yqjG	1.1E-8 ± 9.2E-10a	3.4E-9 ± 2.4E-10a	1.4E-8 ± 1.7E-9a	4.9E-7 ± 2.1E-7	1.0E-7 ± 4.5E-9	1.2E-7 ± 7.0E-8	2.9E-7 ± 9.7E-8	1.4E-7 ± 2.7E-8
yajL	5.6E-8 ± 2.8E-8a	1.4E-8 ± 7.5E-9a	1.0E-7 ± 3.1E-8a	3.7E-7 ± 1.9E-7	8.6E-7 ± 2.8E-7	2.2E-7 ± 1.2E-7	2.5E-7 ± 1.3E-7	4.4E-7 ± 2.9E-7
hemE	8.7E-10 ± 6.1E-11a	6.9E-10 ± 2.3E-11a	5.6E-9 ± 5.3E-10a	1.9E-7 ± 3.4E-8	1.2E-8 ± 7.7E-10	1.2E-7 ± 3.5E-8	1.4E-7 ± 5.4E-8	4.8E-8 ± 5.7E-9
ilvA	1.6E-8 ± 4.1E-9a	2.8E-8 ± 3.6E-8a	5.9E-7 ± 1.7E-7a	2.9E-7 ± 4.1E-8	2.9E-7 ± 5.9E-8	1.5E-6 ± 6.8E-7	8.5E-8 ± 5.4E-8	1.3E-6 ± 5.3E-7
prpD	1.6E-7 ± 1.7E-7a	9.0E-8 ± 1.8E-7a	1.3E-7 ± 2.1E-8a	7.3E-7 ± 1.0E-7	6.2E-7 ± 4.2E-7	7.9E-7 ± 1.7E-7	1.3E-6 ± 8.7E-7	2.4E-6 ± 5.7E-7
zwf	1.4E-8 ± 1.5E-9a	2.3E-8 ± 2.7E-8a	7.2E-8 ± 1.7E-8a	1.2E-6 ± 5.3E-7	5.8E-7 ± 2.2E-7	3.7E-7 ± 2.7E-7	5.6E-7 ± 3.6E-7	6.4E-7 ± 2.7E-7
eda	3.9E-9 ± 2.7E-10a	2.0E-9 ± 2.6E-10a	9.2E-8 ± 1.6E-8a	3.3E-7 ± 1.1E-7	2.6E-7 ± 9.4E-8	2.6E-7 ± 1.1E-7	2.2E-7 ± 2.4E-7	4.6E-7 ± 2.1E-7
gor	2.4E-8 ± 3.5E-9a	1.2E-8 ± 1.8E-9a	8.3E-8 ± 3.4E-8a	1.1E-6 ± 5.5E-7	8.6E-7 ± 5.0E-7	2.2E-7 ± 8.2E-8	9.0E-7 ± 5.0E-7	6.1E-7 ± 3.7E-7
pqqL	5.4E-9 ± 4.7E-10a	1.8E-9 ± 2.3E-10a	6.7E-8 ± 1.1E8a	3.0E-7 ± 2.0E-7	3.0E-7 ± 1.1E-7	3.7E-7 ± 2.7E-7	1.5E-7 ± 9.2E-8	6.3E-7 ± 3.8E-8
a Significant difference to its normal peptide control and fluorescent dye control ( p < 0.05).

Table 4. Top 10 protein list of [SD][QV][AEDT]A[YIL][CE][AK][ARL][MV][AHK]? [KET][LV][AYLF]E enriched in entire E. coli K12. The motif was searched in entire E. coli K12 proteome by GLAM2SCAN.

Rank	Name	EcoGene Accession	START	SITE	END	SCORE
1	ilvA	EG10493	266	DSDAICAAMKDLFE	279	29.2
2	thrS	EG11001	116	DVEALEKRMHELAE	129	28.1
3	yqjG	EG12746	202	SQEAYDEAVAKVFE	215	26
4	pqqL	EG11744	357	MQDAANALMAELAT	370	24.3
5	prpD	EG13603	293	SQTAVEAAM.TLYE	305	23
6	eda	EG10256	53	AVDAIRAIAKEVPE	66	22.6
7	gor	EG10412	87	SRTAYIDRIHTSYE	100	22.5
8	zwf	EG11221	54	DKAAYTKVVREALE	67	21.8
9	yajL	EG13272	101	IVAAICAAPATVLV	114	21.1
10	hemE	EG11543	174	DPQALHALLDKLAK	187	20

Table 5. Secondary structure of Motifs from 10 E. coli K12 identified proteins and human S100A1 proteins. The secondary structure of motifs for each protein was provided by their provider and RCSB PDB with protein 3D structures. However, the hemE, zwf and pqqL do not have the protein 3D structure in RCSB PDB. We used the EcoGene 3.0 which contains the QUARK prediction method to predict the secondary structure of motifs.

Name	Secondary structure of Motif	Source	PDB ID	QUARK ID	Reference
thrS	Alpha-helix	RCSB PDB	1TJE		(34, 39)
yqjG	Alpha-helix	RCSB PDB	4G0L		(38, 39)
yajL	Beta-sheet+Alpha-helix	RCSB PDB	2AB0		(36, 39)
hemE	Alpha-helix	EcoGene 3.0		E11780	(41, 42)
ilvA	Alpha-helix	RCSB PDB	1TDJ		(33, 39)
prpD	Alpha-helix	RCSB PDB	1SZQ		(31, 39)
zwf	Alpha-helix	EcoGene 3.0		E14278	(41, 42)
eda	Alpha-helix	RCSB PDB	1WAU		(37, 39)
gor	Alpha-helix	RCSB PDB	1GEU		(32, 39)
pqqL	Alpha-helix	EcoGene 3.0		E12551	(41, 42)
S100A1	Alpha-helix	RCSB PDB	1ZFS		(35, 39)

Table 6. K_d values for 2-oxohistidine peptides binding to S100A1.

	Oxidative Peptides			Normal Peptides			Fluorescent Dyes
S100A1	DyLight 550 oxo-AG	DyLight 550 oxo-SE	DyLight 650 oxo-IA	DyLight 650 AG	DyLight 650 SE	DyLight 550 IA	DyLightTM 550	DyLightTM 650
w/o calciumb	5.3E-9 ± 4.9E-9a	5.2E-9 ± 2.1E-9a	1.2E-8 ± 4.2E-9a	3.9E-8 ± 4.0E-8	8.2E-8 ± 5.7E-8	3.1E-8 ± 2.0E-8	3.4E-7 ± 2.4E-7	3.6E-7 ± 3.6E-7
w/ calcium	7.3E-9 ± 4.8E-9a	2.9E-9 ± 6.4E-10a	2.8E-8 ± 9.8E-9a	1.3E-7 ± 9.1E-8	8.5E-8 ± 5.5E-8	6.6E-8 ± 3.7E-8	5.6E-8 ± 3.8E-8	1.2E-7 ± 8.4E-8
a Significant difference to its normal peptide control and fluorescent dye control ( p < 0.05). b No significant difference to with calcium group (p > 0.1).

Figures

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Figure 7A.

Figure 7B.

Figure 8.