Comparative Proteomic Profiling of Unannotated Microproteins and Alternative Proteins in Human Cell Lines

Ribosome
profiling and mass spectrometry have revealed thousands
of small and alternative open reading frames (sm/alt-ORFs) that are
translated into polypeptides variously termed as microproteins and
alt-proteins in mammalian cells. Some micro-/alt-proteins exhibit
stress-, cell-type-, and/or tissue-specific expression; understanding
this regulated expression will be critical to elucidating their functions.
While differential translation has been inferred by ribosome profiling,
quantitative mass spectrometry-based proteomics is needed for direct
comparison of microprotein and alt-protein expression between samples
and conditions. However, while label-free quantitative proteomics
has been applied to detect stress-dependent expression of bacterial
microproteins, this approach has not yet been demonstrated for analysis
of differential expression of unannotated ORFs in the more complex
human proteome. Here, we present global micro-/alt-protein quantitation
in two human leukemia cell lines, K562 and MOLT4. We identify 12 unannotated
proteins that are differentially expressed in these cell lines. The
expression of six micro/alt-proteins from cDNA was validated biochemically,
and two were found to localize to the nucleus. Thus, we demonstrate
that label-free comparative proteomics enables quantitation of micro-/alt-protein
expression between human cell lines. We anticipate that this workflow
will enable the discovery of regulated sm/alt-ORF products across
many biological conditions in human cells.

核糖体谱（ribosome profiling）与质谱（mass spectrometry）技术已在哺乳动物细胞中发现了数千个小开放阅读框与可变开放阅读框（sm/alt-ORFs），其翻译产生的多肽分别被称为微蛋白（microproteins）与可变蛋白（alt-proteins）。部分微蛋白/可变蛋白呈现应激、细胞类型特异性以及/或组织特异性表达模式；解析这类受调控的表达机制，对阐明其功能至关重要。尽管核糖体谱技术可推断差异翻译现象，但要直接比较不同样本与实验条件下微蛋白与可变蛋白的表达水平，仍需依赖基于质谱的定量蛋白质组学方法。不过，尽管无标记定量蛋白质组学（label-free quantitative proteomics）已被用于检测细菌微蛋白的应激依赖性表达，但该方法尚未被用于分析更为复杂的人类蛋白质组中未注释开放阅读框的差异表达情况。本研究针对两种人类白血病细胞系K562与MOLT4开展了全范围微蛋白/可变蛋白定量分析，共鉴定出12种在这两种细胞系中存在差异表达的未注释蛋白。通过生化实验验证了6种源自互补DNA（cDNA）的微/可变蛋白的表达情况，并发现其中2种定位于细胞核。综上，本研究证实无标记比较蛋白质组学可用于定量分析人类细胞系间微蛋白/可变蛋白的表达水平。我们预计该分析流程将助力在人类细胞的多种生物学条件下，发现受调控的sm/alt-ORF表达产物。