diff --git a/content/05.protein-extraction.md b/content/05.protein-extraction.md index 9e85ebe7..670636ac 100644 --- a/content/05.protein-extraction.md +++ b/content/05.protein-extraction.md @@ -1,4 +1,4 @@ -## 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. diff --git a/content/06.proteolysis.md b/content/06.proteolysis.md index ba323c19..48c1c36d 100644 --- a/content/06.proteolysis.md +++ b/content/06.proteolysis.md @@ -1,4 +1,4 @@ -## 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. diff --git a/content/07.peptide-quantification.md b/content/07.peptide-quantification.md index b72a56fe..5c523568 100644 --- a/content/07.peptide-quantification.md +++ b/content/07.peptide-quantification.md @@ -1,4 +1,4 @@ -## 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**). diff --git a/content/08.enrichment.md b/content/08.enrichment.md index f1982bb6..13c26157 100644 --- a/content/08.enrichment.md +++ b/content/08.enrichment.md @@ -1,4 +1,4 @@ -## 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 diff --git a/content/09.peptide-purification.md b/content/09.peptide-purification.md index baef71a0..e7bb1e0d 100644 --- a/content/09.peptide-purification.md +++ b/content/09.peptide-purification.md @@ -1,6 +1,8 @@ -## 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. diff --git a/content/10.liquid-chromatography.md b/content/10.liquid-chromatography.md index 645527ea..e703222e 100644 --- a/content/10.liquid-chromatography.md +++ b/content/10.liquid-chromatography.md @@ -1,4 +1,4 @@ -## 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. diff --git a/content/11.peptide-ionization.md b/content/11.peptide-ionization.md index c10c4f5c..3416333c 100644 --- a/content/11.peptide-ionization.md +++ b/content/11.peptide-ionization.md @@ -1,4 +1,5 @@ -## 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]. diff --git a/content/12.mass-spectrometers.md b/content/12.mass-spectrometers.md index 89ab9a5e..d8e6a559 100644 --- a/content/12.mass-spectrometers.md +++ b/content/12.mass-spectrometers.md @@ -1,4 +1,4 @@ -## 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. diff --git a/content/13.Peptide-Fragmentation.md b/content/13.Peptide-Fragmentation.md index 0401ee79..55211b08 100644 --- a/content/13.Peptide-Fragmentation.md +++ b/content/13.Peptide-Fragmentation.md @@ -1,4 +1,4 @@ -## Tandem Mass Spectrometry and Peptide Fragmentation {.page_break_before} +## 11. Tandem Mass Spectrometry and Peptide Fragmentation {.page_break_before} ### Tandem Mass Spectrometry diff --git a/content/14.Data-Acquisition.md b/content/14.Data-Acquisition.md index 527ebc6f..15e12856 100644 --- a/content/14.Data-Acquisition.md +++ b/content/14.Data-Acquisition.md @@ -1,4 +1,4 @@ -## 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. diff --git a/content/15.raw-data-analysis.md b/content/15.raw-data-analysis.md index d0713406..275ff3e7 100644 --- a/content/15.raw-data-analysis.md +++ b/content/15.raw-data-analysis.md @@ -1,4 +1,4 @@ -## 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. diff --git a/content/16.databases.md b/content/16.databases.md index c4a4fc2d..abc7da91 100644 --- a/content/16.databases.md +++ b/content/16.databases.md @@ -1,5 +1,4 @@ -## 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=*. diff --git a/content/17.knowledgebases.md b/content/17.knowledgebases.md index 0a80a670..b28dc954 100644 --- a/content/17.knowledgebases.md +++ b/content/17.knowledgebases.md @@ -1,4 +1,4 @@ -## 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. diff --git a/content/18.biological-interpretation.md b/content/18.biological-interpretation.md index 759fe563..f7374852 100644 --- a/content/18.biological-interpretation.md +++ b/content/18.biological-interpretation.md @@ -1,4 +1,4 @@ -## 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. diff --git a/content/19.validation.md b/content/19.validation.md index 72578b7b..6c001b48 100644 --- a/content/19.validation.md +++ b/content/19.validation.md @@ -1,4 +1,4 @@ -## Orthogonal experimental methods {.page_break_before} +## 17. Orthogonal Validation {.page_break_before} ### The importance of orthogonal experimental validation