Toward a Comprehensive Characterization of a Human Cancer Cell Phosphoproteome

Abstract

Mass spectrometry (MS)-based phosphoproteomics has achieved extraordinary success in qualitative and quantitative analysis of cellular protein phosphorylation. Considering that an estimated level of phosphorylation in a cell is placed at well above 100,000 sites, there is still much room for improvement. Here, we attempt to extend the depth of phosphoproteome coverage while maintaining realistic aspirations in terms of available material, robustness, and instrument running time. We developed three strategies, where each provided a different balance between these three key parameters. The first strategy simply used enrichment by Ti(4+)-IMAC followed by reversed chromatography LC-MS (termed 1D). The second strategy incorporated an additional fractionation step through the use of HILIC (2D). Finally, a third strategy was designed employing first an SCX fractionation, followed by Ti(4+)-IMAC enrichment and additional fractionation by HILIC (3D). A preliminary evaluation was performed on the HeLa cell line. Detecting 3700 phosphopeptides in about 2 h, the 1D strategy was found to be the most sensitive but limited in comprehensivity, mainly due to issues with complexity and dynamic range. Overall, the best balance was achieved using the 2D based strategy, identifying close to 17,000 phosphopeptides with less than 1 mg of material in about 48 h. Subsequently, we confirmed the findings with the K562 cell sample. When sufficient material was available, the 3D strategy increased phosphoproteome allowing over 22,000 unique phosphopeptides to be identified. Unfortunately, the 3D strategy required more time and over 1 mg of material before it started to outperform 2D. Ultimately, combining all strategies, we were able to identify over 16,000 and nearly 24,000 unique phosphorylation sites from the cancer cell lines HeLa and K562, respectively. In summary, we demonstrate the need to carry out extensive fractionation for deep mining of the phosphoproteome and provide a guide for appropriate strategies depending on sample amount and/or analysis time.

Cdk1-mediated phosphorylation of Cdc7 suppresses DNA re-replication

“…Multiple large-scale proteomic analyses have indicated that Cdc7 is phosphorylated at Ser16, Ser302, and Thr503 in prometaphase, [26][27][28] which is consistent with our prediction (Fig 1E.). To further confirm it, we determined the extent of hyper-phosphorylation in vivo by two-dimensional PAGE with mycFLAGtagged Cdc7 WT and Cdc7 £5A expressed in HEK293T cells.…”

“…This may be due to Cdc7 autophosphorylation 14 or phosphorylation by other kinases such as polo-like kinase (Plk), as large scale proteomics studies indicated that Plk phosphorylates Cdc7 at Serine 27 and 277 in prometaphase. 26,28,[30][31][32][33] In any event, our data demonstrate that Cdc7 is phosphorylated at multiple sites in a mitotic Cdkdependent manner around prometaphase.…”

Enrichment Strategies for Identification and Characterization of Phosphoproteome

“…All peptides will bind to a cation exchanger at low pH condition, and bound peptides are predominantly separated by a stepwise elution with increasing ionic strength and/or increasing pH value. 28 Through the above process, the peptides obtained from complex biological sample are divided to 6~12 fractions. Phosphoprotein (or phosphpeptides) bound to SCX matrix are eluted in the early fractions, and peptides except for phosphorylated counterparts are eluted gradually on high salt or pH conditions.…”

“…HILIC has been reported frequently in recent years in phosphoproteomic studies, because highly hydrophilic solutes as phosphorylated ones in comparison with common peptides are retained on the column for the longest time. [28][29][30] In HILIC, hydrophilic components acquired from biological specimens are typically bound onto HILIC column matrix when used with below 10 % aqueous. To isolate the phosphopeptides from ordinary ones, all peptides are fractionated by predominantly increasing the aqueous mobile phase up to 40% (Figure 2-B).…”

“…A variety of enrichment and fractionation methods in phosphoproteomics have been reported including the enrichment with immobilized metal affinity chromatography (IMAC) 6,[14][15][16] and metal oxide affinity chromatography (MOAC) as well as titanium dioxide (TiO 2 ), [17][18][19][20][21][22][23][24][25] and fractionation with strong cation exchange (SCX), 26,27 hydrophilic interaction chromatography (HILIC), [28][29][30] and electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). [31][32][33] More recently, the two sequential steps of preparation methods are commonly used in large scale phosphoproteomics.…”