Systematic assessment of survey scan and MS2-based abundance strategies for label-free quantitative proteomics using high-resolution MS data

Abstract

Survey-scan-based label-free method have shown no compelling benefit over fragment ion (MS2)-based approaches when low-resolution mass spectrometry (MS) was used, the growing prevalence of high-resolution analyzers may have changed the game. This necessitates an updated, comparative investigation of these approaches for data acquired by high-resolution MS. Here, we compared survey scan-based (ion current, IC) and MS2-based abundance features including spectral-count (SpC) and MS2 total-ion-current (MS2-TIC), for quantitative analysis using various high-resolution LC/MS data sets. Key discoveries include: (i) study with seven different biological data sets revealed only IC achieved high reproducibility for lower-abundance proteins; (ii) evaluation with 5-replicate analyses of a yeast sample showed IC provided much higher quantitative precision and lower missing data; (iii) IC, SpC, and MS2-TIC all showed good quantitative linearity (R(2) > 0.99) over a >1000-fold concentration range; (iv) both MS2-TIC and IC showed good linear response to various protein loading amounts but not SpC; (v) quantification using a well-characterized CPTAC data set showed that IC exhibited markedly higher quantitative accuracy, higher sensitivity, and lower false-positives/false-negatives than both SpC and MS2-TIC. Therefore, IC achieved an overall superior performance than the MS2-based strategies in terms of reproducibility, missing data, quantitative dynamic range, quantitative accuracy, and biomarker discovery.

Figure 1

Quantitative reproducibility of the three label-free methods. (A) Comparison of the coefficient-of-determination (R²) of the linear regression by three methods including spectral count (SpC), MS/MS total ion current (MS2-TIC), and ion current (IC). Data of duplicate LC/MS runs of seven types of proteomic samples (human bronchoalveolar lavage fluid, human skeletal muscle cells, rat brain, rat liver, rat retina, E. coli cells, and yeast cells) were analyzed, and each data point represents the R² of one of the proteome samples. The high-abundance proteins refer to the top 33% of all proteins ranked by spectral count, and the rest are designated as lower-abundance proteins. (B) Representative scatter plots of duplicate LC-MS/MS analyses by SpC, MS2-TIC, and IC. The two axes represent the quantitative abundance values of the same proteins, respectively, by the two duplicate runs.

Figure 2

Coefficients of variation (CV) of the abundance values of the 1196 quantified yeast proteins by SpC, MS2-TIC, and IC (N = 5 LC-MS analyses). (A) Box-and-whisker plot analysis was employed to show the spread of protein CVs around the median value (the horizontal line inside the box); bottom and top of the boxes correspond to the top 25th and 75th percentile of the CV distribution and whiskers to the minimum and maximum values. (B) The distribution of CV vs protein abundance. Red circles indicate SpC, black squares indicate MS2-TIC, and blue triangles indicate IC data spot.

Figure 3

Quantitative responses of spectral count (SpC), MS2-TIC and ion current (IC) vs protein abundance levels. BSA was spiked into E. coli extract at six different levels spanning a concentration range >1000. Excellent linearity was observed for (A) spectral count (SpC), (B) MS2-TIC, and (C) ion current (IC). As no BSA-derived peptide was identified in the lowest level, the level was below the detection limits of SpC and MS2-TIC; by comparison, this level can be quantified by IC with sufficient S/N.

Figure 4

Linear regression analysis correlating the quantitative values with protein loading amounts, by spectral count (SpC), MS2-TIC, and ion current (IC). The quantitative values of individual proteins in 1, 2, and 4 μg loading were individually plotted against these with 0.5 μg. Slopes of trend lines and R² values are shown.

Figure 5

Distribution of the protein ratios in a CPTAC data set (Study 6B over 6A) for (A) the 14 UPS proteins and (B) 761 yeast proteins quantified by spectral count (SpC), MS2-TIC, and ion current (IC).

Figure 6

Volcano plots illustrating the discovery of altered proteins in CPTAC study 6B vs 6A set by spectral count (SpC, panels A and B), MS2-TIC (C and D), and ion current (IC, E and F) approaches. The levels of the 14 UPS proteins are different between the two groups (nominal 6B/6A ratio ≈ 3), whereas the levels of yeast proteins are the same. The Y-axis shows the log2 ratios of proteins quantified, and the X-axis shows the p-values (by Student’s t-test) for the comparison. Each dot represents a unique protein group, and the dashed lines denote the cutoff thresholds (p ≤ 0.05 and >2-fold change) that define significantly altered proteins, which are in turn shown as red dots.