Quantitative Profiling of Post-translational Modifications by Immunoaffinity Enrichment and LC-MS/MS in Cancer Serum without Immunodepletion

Abstract

Biomarker identification is a key step for illustration of disease mechanisms, drug development, and diagnostics. Diagnostics research has focused on identifying biomarkers from viable biofluids, including serum/plasma, saliva, cerebrospinal fluid, and urine. Due to ease of collection and richness in proteins and metabolites, serum/plasma has been the preferred choice for diagnostic studies (1-3). Advancement of mass-spectrometry-based proteomic technologies has allowed identification and quantification of thousands of proteins in serum/plasma samples. Typically, these methods combine isotopic labeling, offline fractionation, and LC-MS/MS analysis. Facilitated by high-throughput proteomics analysis, researchers have collected vast amounts of comparative information about protein abundance in serum/plasma of patients of various types of diseases that accelerated the identification of potential biomarkers (4).To date, the majority of serum/plasma proteomic work has been conducted to analyze total protein level abundance, with only a few studies to analyze posttranslational modifications (PTMs) 1 , usually glycosylation (5, 6). As one of the most important mechanisms for regulating protein function, PTMs, including phosphorylation, acetylation, ubiquitination, and methylation, have been identified and validated as critical for signaling transduction, protein degradation, and transcriptional regulation (7,8). Currently, there exists very limited data about PTMs in serum/plasma beyond glycosylation. The abundant serum protein albumin has long been known to be acetylated by aspirin, and this reaction can occur in vitro without the presence of any acetyltransferase (9). Fibrinogen, another abundant serum protein, is also acetylated by aspirin both in vivo and in vitro (10, 11). These previous findings and the known importance of PTMs in cellular signaling provided the impetus for a large-scale survey of PTMs other than glycosylation by immunoaffinity enrichment of PTM-containing peptides.One challenge for proteomic analysis of serum/plasma is the broad dynamic range of the serum/plasma proteome (12), including a high percentage of the total protein content of serum/plasma represented by only 12 proteins. This limitation can be partially overcome by immunodepletion of abundant proteins prior to enzymatic digestion (4, 13), however, generation of the large quantities of materials necessary for PTM enrichment with an immunodepletion workflow could be cost prohibitive. It was therefore of interest to develop a PTM

Omics technologies in diagnostics of lung adenocarcinoma

“…Gu и соавт. провели [44] сравнительный протеомный анализ сыворотки крови у больных раком лёгких (АКЛ в трёх случаях из четырех), раком молочной железы и острым миелоидным лейкозом (AML). Используя методы иммуноаффинного обогащения и обогащения пептидов на IMAC-Fe 3+ с последующей масс-спектрометрической идентификацией и безметковой количественной оценкой по XIC, они идентифицировали 431 пептид с ацетилированными остатками лизина, 138 пептидов с монометилированными остатками аргинина и 148 пептидов с метилированными остатками лизина в образцах НМКРЛ.…”

“…Для монометилированного по аргинину белка ARID1A (R1593) уровень модифицированного пептида был ниже в случае НМКРЛ. Кроме того, ацетилированные белки альбумин, APOA1, серотрансферрин (TF) и альфа-2-макроглобулин достоверно различались в случае аденокарциномы лёгкого и плоскоклеточного рака лёгких [44]. 5.…”

A Phosphoproteomic Screen Identifies a Guanine Nucleotide Exchange Factor for Rab3A Protein as a Mitogen-activated Protein (MAP) Kinase Phosphatase-5-regulated MAP Kinase Target in Interleukin 6 (IL-6) Secretion and Myogenesis

“…Phospho-MAPK Substrate Proteomic Screen-The PhosphoScan method was performed as described previously using services provided by Cell Signaling Technology (11)(12)(13). Nondamaged and damaged skeletal muscle from mkp-5 ϩ/ϩ and mkp-5 Ϫ/Ϫ mice were homogenized, sonicated, and clarified by centrifugation at 20,800 ϫ g for 20 min at 4°C.…”

Proteomics of the Salivary Fluid

“…Proteomics is not only addressing the efficient separation of peptides upon, mostly, tryptic digestion of proteins and their sensitive detection using mass spectrometry. Proteomics is a technology enabling significantly and profoundly better approach to investigating and understanding proteins' function and their posttranslational modifications [7][8][9][10][11][12]. Applying proteomics methods for analysis of clinical samples is especially important in time of personalized medicine, which tailors individualized treatment of each patient based on specification of the diseases [3,[13][14][15][16][17][18][19].…”