add section numbers

content/05.protein-extraction.md

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-## Protein Extraction {.page_break_before}
+## 3. Protein Extraction {.page_break_before}
 
 Protein extraction is the initial phase of any mass spectrometry-based proteomics experiment.
 Protein extraction is sample dependent; a solution that is effective for plasma proteomics may not work well for plant tissue proteomics. 

content/06.proteolysis.md

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-## 6. Proteolysis {.page_break_before}
+## 4. Proteolysis {.page_break_before}
 Proteolysis is the defining step that differentiates bottom-up or shotgun proteomics from top-down proteomics. 
 Hydrolysis of proteins is extremely important because it defines the population of potentially identifiable peptides. 
 Generally, peptides between a length of 7-35 amino acids are considered useful for mass spectrometry analysis.

content/07.peptide-quantification.md

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-## Mass Spectrometry Methods for Peptide Quantification {.page_break_before}
+## 5. Peptide Quantification {.page_break_before}
 
 ### Label-free quantification (LFQ) of peptides
 LFQ of peptide precursors requires no additional steps in the protein extraction, digestion, and peptide purification workflow (**Figure 3**). 

content/08.enrichment.md

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-## Peptide/Protein Enrichment and Depletion {.page_break_before}
+## 6. Enrichment and Depletion {.page_break_before}
 In order to study low abundance protein modifications, or to study rare proteins in complex mixtures, various methods have been developed to enrich or deplete specific proteins or peptides. 
 
 ### Peptide enrichment 

content/09.peptide-purification.md

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-## Peptide Purification and Fractionation {.page_break_before}
+## 7. Peptide Purification and Fractionation {.page_break_before}
 
 ### Peptide purification methods
+Before peptide analysis, interferences from sample preparation must be removed. 
+There are several approaches to purify peptides. 
 
 #### Solid phase extraction (SPE)
 Solid phase extraction (SPE) is a common MS-based proteomics technique employed in the sample preparation. 

content/10.liquid-chromatography.md

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-## Liquid Chromatography (LC) {.page_break_before}
+## 8. Liquid Chromatography (LC) {.page_break_before}
 
 Chromatography is the physical sorting of a mixture of molecular species that are dissolved in a mobile phase through the strength of binding, or affinity, to the chromatographic column’s stationary phase [@DOI:10.1016/j.aca.2009.03.041]. 
 The mobile phase is pressure driven through the column and molecular species, or analytes, that have a strong affinity to the stationary phase are retained, or slowed, while those with a weak affinity pass through quickly. 

content/11.peptide-ionization.md

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-## Peptide Ionization {.page_break_before}
+## 9. Peptide Ionization {.page_break_before}
+
 As early as the late 1950s, derivitization reagents were used to make peptides volatile enough for electron impact ionization analysis [@DOI:10.1021/ja01518a069]. 
 Eventually this led to GC-MS analysis of derivatized peptides for sequencing [@DOI:10.1021/ja00802a048].
 In the early 1980s, fast atom bombardment (FAB) enabled peptide ionization and sequencing by MS/MS [@DOI:10.1021/ac00234a035], but difficulty interfacing FAB with LC limited its utility [@DOI:10.1021/ja3094313].

content/12.mass-spectrometers.md

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-## Types of Mass Spectrometers used for Proteomics {.page_break_before}
+## 10. Mass Spectrometers {.page_break_before}
 ### Mass spectrometry
 Mass spectrometry is a science of ions; mass spectrometers serve as sophisticated instruments for determining the masses of compounds and elements. 
 Mass spectrometers can therefore be likened to an ultra-precise weigh scale that can differentiate mass variations down to a single electron, or even lighter. 

content/13.Peptide-Fragmentation.md

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-## Tandem Mass Spectrometry and Peptide Fragmentation {.page_break_before}
+## 11. Tandem Mass Spectrometry and Peptide Fragmentation {.page_break_before}
 
 ### Tandem Mass Spectrometry
 

content/14.Data-Acquisition.md

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-## Data Acquisition {.page_break_before}
+## 12. Data Acquisition {.page_break_before}
 
 Hybrid mass spectrometers used for modern proteome analysis offer the flexibility to collect data in many different ways. 
 Data acquisition strategies differ in the sequence of precursor scans and fragment ion scans, and in how analytes are chosen for MS/MS. 

content/15.raw-data-analysis.md

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-## Analysis of Raw Data {.page_break_before}
+## 13. Raw Data Analysis {.page_break_before}
 
 The goal of raw data analysis is to convert raw spectral data into lists of altered protein groups, which requires many steps, including checking data quality, peptide spectra matching, protein inference [@PMID:16009968; @DOI:10.1016/j.jprot.2016.08.002], quantification, and statistical hypothesis tests. 
 Subsequently, many additional analyses can be performed to make biological inferences, which is covered in a subsequent section. 

content/16.databases.md

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-## Protein Sequence Databases {.page_break_before}
-
+## 14. Protein Sequence Databases {.page_break_before}
 
 ### Where do you get them?
 For those looking for guidance on where to obtain a database for their organism of interest quickly, we recommend going to uniprot.org and using their "proteomes": https://www.uniprot.org/proteomes?query=*. 

content/17.knowledgebases.md

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-## Proteomics Data Repositories and Knowledge Bases {.page_break_before}
+## 15. Data Repositories and Knowledge Bases {.page_break_before}
 
 ### Proteomics raw data repositories
 An essential part of the proteomics publication cycle is raw data sharing. 

content/18.biological-interpretation.md

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-## Biological Interpretation  {.page_break_before}
+## 16. Biological Interpretation  {.page_break_before}
 
 The most common untargeted proteomics experiment will produce a list of proteins or peptides of interest which require further validation and biological interpretation.
 This list usually results from statistical data analysis; the typical output of differentially expressed proteins usually contains hundreds of hits. 

content/19.validation.md

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-## Orthogonal experimental methods {.page_break_before}
+## 17. Orthogonal Validation {.page_break_before}
 
 ### The importance of orthogonal experimental validation