A Multipathway Phosphopeptide Standard for Rapid Phosphoproteomics Assay Development

Abstract

Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin-like growth factor-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564.

Keywords: data-independent acquisition; mass spectrometry; phosphopeptide; phosphorylation; proteomics; stable isotope label; targeted.

Graphical abstract

Fig. 1

Peptide properties for the multi-pathway phosphopeptide standard.A, pie chart showing the relative breakdown of selected phosphopeptides across signaling pathways. Histograms showing (B) the number of observations for each peptide in the Phosphopedia database (log₁₀ scale), (C) the distribution of peptide lengths, and (D) the distribution of estimated iRT values. iRT, indexed retention time.

Fig. 2

Experimental design.A, a schematic showing key steps in our experimental approach to assess the lab-to-lab variability of the multipathway phosphopeptide standard. Key steps, such as cell culture, digestion, mixing, and data analysis were controlled, while variability from IMAC enrichment and mass spectrometry instrumentation was isolated in each lab site. B, all lab sites were instructed to use the same eight-injection GPF-DIA method to ensure consistency between lab sites. In this approach, eight injections of the same sample were made, spanning 100 m/z ranges. Each injection was performed using staggered windowing to achieve 2 m/z precursor isolation (targeted PRM equivalent isolation). DIA, data-independent acquisition; GPF, gas-phase fractionation; IMAC, immobilized metal ion affinity chromatography; PRM, parallel reaction monitoring.

Fig. 3

Measurement consistency across labs.A, the total number of observed heavy and endogenous peptides at each lab site. For each peptide, the heavy form must be confidently detected (correct retention time, fragmentation, and mass accuracy) for the corresponding endogenous peptide to be considered “observed”, ensuring a light/heavy ratio. Similarly, endogenous peptides were only “observed” if they had light/heavy ratios >1/100 to protect against integrating noise. B, a violin plot showing the CV between measurements across labs for the top 30 peptides (first two orders of magnitude in ratio) using either total intensity or the light/heavy ratio for both MS1 and MS2 data. Black boxes indicate the interquartile range, while the white points indicate the median CV.

Fig. 4

Peptide quantification accuracy across sites.Box plots show the median and estimated quartiles of log₁₀ normalized light/heavy ratios for confidently observed heavy peptides at each lab site. Whiskers indicate the full range of values, while gray dots indicate the actual ratios for each site. Individual light intensity values in the pink shaded region (with below 1/100 light/heavy ratio) are considered low confidence and marked as “unobserved” in Figure 3. In addition to the sequence, protein, and site, the number of lab sites that confidently observed each heavy peptide (of five total lab sites) is also indicated. Peptides are sorted by the median light/heavy ratio.

Fig. 5

Comparison of MS1- and MS2-level quantification. Phosphopeptide MS1-level (A) and MS2-level light/heavy ratios (B), as well as MS1-level (C) and MS2-level total light intensities (D) for lab sites B, C, D, and E relative to A. Dashed lines are shown to indicate matching 1:1 agreement between lab-specific measurements. Peptide ratio of ratios that fit closer to the dashed line show lower variability between labs. All axes were selected to show approximately six orders of magnitude.

Fig. 6

Challenges of measuring endogenous AKT1 S473 with wide DIA windows. The peptide RPHFPQFpSYSASGTA produces several fragment ions (A), producing a long consecutive b-ion ladder from b3 to b7 (B), but few y-type ions, as demonstrated by the library entry spectrum (C). The lack of fragment ions containing the heavy-labeled C-terminal alanine residue (light blue) means that these ions must be quantified either from precursors or by separating the light (578.256 m/z) and heavy (579.592 m/z) forms into different precursor isolation windows. D, staggering 4 m/z windows to achieve 2 m/z isolation, as performed by lab sites A, B, C, and E can separate light and heavy integrations. However, normal 4 m/z windows, as performed by lab site D, cofragment light and heavy peptides such that their b-ion signals cannot be separated. DIA, data-independent acquisition.

Fig. 7

Quantifying phosphopeptide positional isomers. Relative light/heavy ratios for five positional isomer pairs in the phosphopeptide mixture (A). Fragment ion chromatograms for positional isomers in the RAF1 peptide SHSESASPSALSSSPNNLSPTGWSQPK (B). Here, S289 and S296 (shaded in gray boxes) are indicated by heavy fragment ion signals, while a third unknown positional isomer of this peptide does not time align with either site.