Fig.1 SEM images of E. coli(A, B) and B. subtilis(C, D) with different magnifications

Fig.2 Investigation of protein coverage and detection reproducibility（A） Numbers of proteins or protein peaks detected from E. coli by MALDI fingerprinting（MALDI）， top-down proteomics（TDP） and bottom-up proteomics（BUP）； the average number was displayed for each approach； （B） numbers of proteins or protein peaks detected from B. subtilis by using the three protein profiling approaches； （C） detection reproducibility for nine repetition tests of E. coli by using MALDI approach； （D） detection reproducibility for nine repetition tests of B. subtilis by using MALDI approach； （E） detection reproducibility for four repetition tests of E. coli by using TDP approach； （F） detection reproducibility for four repetition tests of B. subtilis by using TDP approach； （G） detection reproducibility for four repetition tests of E. coli by using BUP approach； （H） detection reproducibility for four repetition tests of B. subtilis by using BUP approach.

Fig.3 Correlation between MALDI and TDP protein profiles（A） MALDI protein fingerprints of E. coli and B. subtilis， with protein identity labeled on the main peaks. The protein identity was tentatively assigned through matching the fingerprint peak masses to the protein（or protein fragment） masses measured by TDP； insert： fingerprint peak numbers detected from E. coli and B. subtilis with protein identity assigned； （B） investigation of the properties of proteins assigned on the MALDI fingerprints of E. coli and B. subtilis. The investigated properties include isoelectric point（PI）， instability index（II）， aliphatic index（AI）， grand average of hydropathy（GRAVY）， and if the proteins were ribosomal proteins； （C） biological functional analysis of the proteins assigned for E. coli using ClueGO system； （D） biological functional analysis of the proteins assigned for B. subtilis using ClueGO system； gene ontology（GO） terms are represented as nodes， with node size representing the enrichment significance； only the most significant terms were labeled， with the exhibition of representative enriched pathway（P < 0.05） interactions among the main targets； （E） a deep hierarchical clustering analysis of the MALDI fingerprints using cosine correlation distance function and average linkage， correlated with the peak presence profile. The peaks exclusively detected from E. coli and B. subtilis were listed.

Fig.4 Protein preference of TDP and BUP revealed by GO enrichment analysis

Fig.5 BUP profile exhibited the largest overlap between bacterial species（A） Counts of species-common and species-specific proteins or protein peaks detected from E. coli and B. subtilis using MALDI， TDP and BUP respectively； （B） the protein-protein interaction network of the BUP species-common proteins， constructed using the String database and Cytoscape with an indegree score threshold of 30； （C—E） top ten significantly enriched biological processes（BPs） from the species-common proteins（C）， E. coli-specific proteins（D） and B. subtilis-specific proteins（E） detected by BUP； （F—H） top ten significantly enriched molecular functions（MFs） from the species-common proteins（F）， E. coli-specific proteins（G） and B. subtilis-specific proteins（H） detected by BUP. P-value lower than 0.05 indicated a significant enrichment.