enhancing precursor and product ion alignment of chimeric spectra · 2015-05-29 · where mr,pc is...

1
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2015 Waters Corporation y1 y5 y3 y6 y2 y4 y10 y11 y12 y7 y8 y9 y13 b 1 b 2 b 3 b 4 b 7 b 6 b 12 b 8 b 10 b 11 b 9 INTRODUCTION Given, the 1. limited number of elements comprising amino acids 1 , 2. similarity in amino acid distribution between proteins, 3. number of enzymatically produced peptides, 4. varying charge-states and isotopes (both precursors and products) The number of ions that can be sampled and identified relative to the total number ions present 2 per unit time is limited regardless of acquisition method Acquisition strategies employing faster acquisition times, including narrow mass isolation widths, have been employed increasing the number of ions sample/unit time. However, faster acquisition times sample less of the peak volume limiting sensitivity. SWATH employs wider mass isolation widths, at somewhat slower scan speeds, increasing both sensitivity as well as the number of ions sampled per unit time albeit, the resultant product ion spectra are highly chimeric. MS E and HDMS E sample all precursor ions with product ions aligned by center mass, retention-time (MS E ) or retention- and drift-time (HDMS E ). However, as with SWATH the product ion spectra are highly chimeric limiting dynamic range. Often it is a plurality (>1) of peptides that best describe an ms/ms spectra in a DIA acquisition. Described herein is a Relative Defect Ion Filter, a de-novo sequencing algorithm capable of identifying multiply peptides in a single Product Ion Spectra. Enhancing Precursor and Product Ion Alignment of Chimeric Spectra Roy Martin; Steve Ciavarini; Scott Geromanos Waters Corporation, Milford, MA Fig- ure 1. Low & Elevated Energy MS E spectra. We will illustrate RDIF processing of the 790.3994, 599.3187 & 484.7480 2 + A 0 pre- cursor ions representing a high, medium and low intensity sampling. Nine of the ten most intense precursor ions were identified with 3 of the 9 being identified to two peptides each. The results illustrate not only multiple peptides but multiple peptides to what would have been perceived to be a single precursor ion. METHODS Sample: Tryptically digested lysates of HeLa, MDA-MB-231 and Yeast. LC: Waters nanoAcquity UPLC with a 75mm X 25cm BEH C18 packed with 1.7mm particles. A = Aqueous 0.2% FA, B = Acn 0.25% FA Gradient: 5-35% B in 60 minutes, () nl/min, X° C Mass Analyzer: Waters SYNAPT G2-Si. 25K mass resolution Acquisition Methods: (HD)MS E , (HD)-DDA, PID Product ions were assigned to each charge-cluster using the described Precursor/Product ion alignment algorithm as DISCUSSION WORKFLOWS (CONT) RESULTS Elevated-Energy Spectrum from Figure 1 8331 Product Post Intensity, m/ z Filtering 371 Product Ions 32 Ions 16 Compliment Pairs Figure 4A D C B Post Filtering 0 Figure 5a Illustrates an extension of ymax by the (m/z)/z associated with valine (99.06, 3.64 ppm). The comple- ment b ion to 1480.7228 is 100.0625. Figure 5b clearly illustrates the absence of the complement. The algorithm cre- ates a virtualion as illustrated in Fig- ure 5b. Figure 5c. Illustrates an isoleu- cine/leucine (m/z)/z (113.08) extension off the Virtualcomplement illustrated in Figure 5b. The 213.16 ion is annotat- ed b 2 and all its attributes are recorded =99.06 0 =113.08 Figure 5a Figure 5b Figure 5c The complement yto the b 2 ion of m/z 213.16 is 1367.6366 which is also a single amino acid extension of (m/z)/z 113.08, isoleucine/leucine off ymax –1 , 1480.7228 as illustrated in Figure 5a. The 1367.6366 ion is annotated ymax –1 and its attributes are recorded. The complement to 1367.63 is then calculat- ed, becoming the new anchor ion (AI) and the process continues. virtualb 1 , 100.0625 b 2 Starting Extend by One from ymax 0 WORKFLOWS 1 2 2 3 3 1 2 4 Compliment Pairs Extension m/z (AA 1-j ) Table 1a Table 1b Table 1c 1 2 3 3 4 5 5 4 4 5 b 5 Figure 6a & 6b Illustrate respectively the post RDIF processing product ions in Table 3 by Ion and Fragment Type. Figure 6b illustrates compliment pairs (red) the number associated to each line connections refers to the m/z pair illustrated in Table 1b. The blue lines represent the product ions by exten- sion. Figure 6b illustrates these same product ions by fragment type (y”, b) also illustrated are the virtualby ex- tension product ions Given Post precursor and product ion alignment, we have a list of precursor- product ions comprising: Precursor isotope charge cluster at charge state z pc and monoisotopic mass A 0 (m/z pc ) Precursor Residual monoisotopic mass A 0 ; i.e., unfragmented precursor found in EE (if found) Filtered Product Ion spectra: list of EE product ions filtered by intensity and m/z Anchor Ion table pre-populated with calculated values for: ymax , b max , y1 (K) , y1 (R) Find Anchor Ions 1. Search the product ions in the Filtered Product Ion Table to find anchor ions: 1.1 If a match is found, then: 1.1.1 Mark this ion as matchedFind Complementary Pairs 1. Calculate the precursor protonated mass: [M r,pc +z pc H] z+ = (m/z pc m p ) * z pc + z * m p where M r,pc is the relative molecular mass of the precursor, m/z pc is the monoisotopic mass of the precursor charge cluster A0 isotope, z pc is the charge state of the precursor, m p is the mass of a proton 2. For each ion (i) in the filtered product spectrum, do: 2.1 For each possible product ion charge state: z p = 1,2,…,(z pc -1), do: 2.1.1 Compute the product ion (i) protonated mass: [M r,i +z i H] z+ at charge state z p [M r,i +z i H] z+ = m/z i * z p 2.1.2 Compute the complementary ion protonated mass [M r,c +z c H] z+ from the precursor: [M r,c +z c H] z+ = [M r,pc +z pc H] z+ - [M r,i +z i H] z+ where M r,c is the relative molecular mass of the complementary product ion and M r,c + M r,i = M r,pc by definition. 2.1.3 Reduce the complementary protonated mass [M r,c +z c H] z+ to the target ion mass m/z c at the complementary charge state z c = z pc - z p : m/z c = [M r,c +z c H] z+ / z c 2.1.4 Search all the other ions in the filtered product spectrum for a complementary match to m/z c (within +/- ppm tolerance): 2.1.4.1 If a match is found, then 2.1.4.1.1 annotate the charge state of this ion: z z c 2.1.4.1.2 mark it as matched2.1.4.1.3 add it to the list of anchor ions. 2.1.4.1.4 Stop the search. Identify Remaining Product Ions—Use Anchors & C-Pairs to Extend Peptide Sequence by 1 Amino Acid 1. For each anchor ion AI i in the Anchor Ion Table (includes Complements & on-the-fly Virtuals”), starting in the order of: ymax , b max , y1 (K) , y1 (R) do: 1.1. For each amino acid AA j in the Amino Acid Table (Table 1c.) do: 1.1.1 Start a new sequence chain n: (denotes a particular precursor/sequence) 1.1.2 Set direction sense: -1 for y-type, +1 for b-type 1.1.3 Calculate extend-by-one target mass m/z T : m/z T = m/z i + direction * m/z j / z i 1.1.4 Search unmatched filtered product ions for a target match (+/- ppm tolerance): 1.1.4.1 If a match is found, then: 1.1.4.1.1 Annotate this product ions properties accordingly: z z i (charge state of the anchor ion) Frag type same as AI i AA type same as AA j chain number n (relates to the number of AAj matching in one loop) 1.1.4.1.2 Mark this ion as matched 1.1.4.1.3 Calculate the Complement and look for a match 1.1.4.1.4 If a match is found, then: 1.1.4.1.4.1 Annotate ion as per 1.1.4.1.1 Else 1.1.4.1.4.2 Create on-the-fly virtual1.1.4.1.4.3 Make AI i = on-the-fly virtual” (m/z VC ) 1.1.4.1.4.4 switch Ion Type (from yto b) 1.1.4.1.4.5 Continue to step 1 1.1.2.2 At completion, IF Match Count = 0 then, Else GoTo 1. Create virtualwhere m/z T = m/z VT = m/z i - m/z j / z i and that becomes the new anchor) 2. Transfer matchedproduct ions from step 1 to Final Product Ion Table 3. For each product ion in Final Product Ion Table, do: 3.1 Query for isotopes using A 0 intensity, z, and delta m/z 3.2 Check Glyco. Status 3.2.1 If Glyco Status = yes”, then 1. where AA j = Glyco Table (Table 3) 3.3 Check Isobaric Labels (TMT, iTRAQ) 3.3.1 If a match is found, then 3.3.1.1 Get reference m/z and Quantify 3.4 Check SILAC Status 3.4.1 If SILAC Status = yes”, then 3.4.1.1 Match SILAC pairs 3.4.1.2 Annotate ion as SILAC pair per 1.1.4.1. 3.4.1.3 Quantify CONCLUSION Most if not all DIA Product Ion Spectra are Chimeric Product Ions to Co-Eluting/Co-Fragmenting Precursors can be Identified and Correctly Quantified provided some continuity in their fragmentation patterns exist Product Ion Mass Accuracy is paramount in grouping product ions to their respective parent precursor in chimeric product ion spectra Often it is a plurality of peptides that best represent a composite DIA product ion spectra Figure 3. Figure 2b. Figure 2a. The six most intense precursor ions illustrated in Figure 1 were processed using the described workflow. Figures 2a & 2b illus- trate flow diagrams relating to respectively, initial product ion filtering and complementary pair generation. Figure 3 repre- sents another flow diagram describing the process of extend- ing Anchor Ions (ymax & b max , Compliments, y1 , Virtual & Vir- tual Complements) one m/z at a time. A m/z can be any user-defined value. As examples Table 1c illustrates the typical m/z values for tryptic peptide where Table 2 illustrates the those used in calculating glycan structures. With respect to quantification the described workflow as illustrated can quanti- fy samples utilizing either isobaric (TMT/iTRAQ) or isotopic SI- LAC labelled samples. For TMT/iTRAQ the user provides the reference product ion m/z in the initial setup. Table 2 Figure 4A-D illustrate the initial process of enhancing precursor and product ion alignment. To begin, the mass analyzer detected 8331 product ions above the LOD. Filtering by m/z and Intensity (<ymax , < Intensity/250) reduced the count to 371 (4.2%), compliment pairs to 32 Ions, 16 complement pairs. Table 3 References 1. Geromanos, S. J., Hughes, C., Golick, D., Ciavarini, S., Gorenstein, M. V., Richardson, K., Hoyes, J. B., Vissers, J. P.C. and Langridge, J. I. (2011), Simulating and validating proteomics data and search results. Proteomics, 11: 11891211. doi: 10.1002/pmic.201000576 2. Geromanos SJ, Hughes C, Ciavarini S, Vissers JP, Langridge JI. Using ion purity scores for enhancing quantitative accuracy and precision in complex proteomics samples. Anal Bioanal Chem. 2012 Sep;404 (4):1127-39. doi:10.1007/s00216-012-6197-y. Epub 2012 Jul 19. PubMed PMID: 22811061. 3. Senko et al, J Am Soc MS 1995 pp. 229-233 Two of a series TP-156, WP-325, MP-435, TP-544 599.3190, 2 + 599.3190, 2 + 790.3990, 2 + 484.7469, 2 + Figure 6a Figure 6a Figure7A B C y9 y8 5x * y1 y1 y10 y9 * In Figure 1A, 9 of the top 10 precursor ions (by intensity) were iden- tified by RDIF processing. Due to space limitations presented is a high, middle and low intensity precursor. ~33% resulted in more than one peptide assignment. Figure 7 panels A & B represent 2- chains off the 599.3190, 2 + precursor. Panel A, illustrates a peptide from G3P1_YEAST, M r 1196.6190. RDIF processing uncovered a sec- ond chain, Panel B, identifying a second peptide from GID3_YEAST, M r 1196.6077. (~10ppm). 240K mass resolving power would be re- quired to separate both at the baseline. The variation in fragment intensity of the y7 & y8 ions in Panel B relate to the 799.3985 ion also being the b 7 (Table 3) of the 790.3990, from YEAST_PGK while y8 is also the 1 + of the 484.7469, 2 + from YEAST_ADH1. y6 (X-P) y7 y6 y5 y4 y3 y2 y1 b 1 b 2 b 3 b 5 b 4 b 7 b 6 b 1 b 2 b 3 b 5 b 8 b 7 b 6 b 4 b 9 b 1 b 2 b 3 b 5 b 4 b 6 b 8 b 7 b 9 y4 y3 y2 y4 y5 y6 ** y7

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Page 1: Enhancing Precursor and Product Ion Alignment of Chimeric Spectra · 2015-05-29 · where Mr,pc is the relative molecular mass of the precursor, m/zpc is the monoisotopic mass of

TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2015 Waters Corporation

y”1 y”5 y”3

y”6 y”2

y”4

y”10 y”11 y”12

y”7

y”8

y”9

y”13

b1 b2 b3 b4 b7

b6

b12 b8 b10

b11 b9

INTRODUCTION

Given, the 1. limited number of elements comprising amino acids1, 2. similarity in amino acid distribution between proteins, 3. number of enzymatically produced peptides, 4. varying charge-states and isotopes (both precursors and

products) The number of ions that can be sampled and identified relative to the total number ions present2 per unit time is limited regardless of acquisition method Acquisition strategies employing faster acquisition times, including narrow mass isolation widths, have been employed increasing the number of ions sample/unit time. However, faster acquisition times sample less of the peak volume limiting sensitivity. SWATH employs wider mass isolation widths, at somewhat slower scan speeds, increasing both sensitivity as well as the number of ions sampled per unit time albeit, the resultant product ion spectra are highly chimeric. MSE and HDMSE sample all precursor ions with product ions aligned by center mass, retention-time (MSE) or retention- and drift-time (HDMSE). However, as with SWATH the product ion spectra are highly chimeric limiting dynamic range. Often it is a plurality (>1) of peptides that best describe an ms/ms spectra in a DIA acquisition. Described herein is a Relative Defect Ion Filter, a de-novo sequencing algorithm capable of identifying multiply peptides in a single Product Ion Spectra.

Enhancing Precursor and Product Ion Alignment of Chimeric Spectra

Roy Martin; Steve Ciavarini; Scott Geromanos Waters Corporation, Milford, MA

Fig-

ure 1. Low & Elevated Energy MSE spectra. We will illustrate RDIF processing of the 790.3994, 599.3187 & 484.7480 2+ A0 pre-cursor ions representing a high, medium and low intensity sampling. Nine of the ten most intense precursor ions were identified with 3 of the 9 being identified to two peptides each. The results illustrate not only multiple peptides but multiple peptides to what would have been perceived to be a single precursor ion.

METHODS

Sample: Tryptically digested lysates of HeLa, MDA-MB-231 and Yeast.

LC: Waters nanoAcquity UPLC with a 75mm X 25cm BEH C18 packed with 1.7mm particles. A = Aqueous 0.2% FA, B = Acn 0.25% FA Gradient: 5-35% B in 60 minutes, () nl/min, X° C

Mass Analyzer: Waters SYNAPT G2-Si. 25K mass resolution

Acquisition Methods: (HD)MSE, (HD)-DDA, PID Product ions were assigned to each charge-cluster using the described Precursor/Product ion alignment algorithm as

DISCUSSION

WORKFLOWS (CON’T) RESULTS

Elevated-Energy Spectrum from Figure 1

8331 Product

Post Intensity, m/z Filtering

371 Product Ions

32 Ions 16 Compliment

Pairs

Figure 4A

D

C

B

Post Filtering

0

Figure 5a Illustrates an extension of y”max by the (m/z)/z associated with valine (99.06, 3.64 ppm). The comple-ment b ion to 1480.7228 is 100.0625. Figure 5b clearly illustrates the absence of the complement. The algorithm cre-ates a “virtual” ion as illustrated in Fig-ure 5b. Figure 5c. Illustrates an isoleu-cine/leucine (m/z)/z (113.08) extension off the “Virtual” complement illustrated in Figure 5b. The 213.16 ion is annotat-ed b2 and all its attributes are recorded

=99.06

0

=113.08

Figure 5a

Figure 5b

Figure 5c

The complement y” to the b2 ion of m/z 213.16 is 1367.6366 which is also a single amino acid extension of (m/z)/z 113.08, isoleucine/leucine off y”max –1, 1480.7228 as illustrated in Figure 5a. The 1367.6366 ion is annotated y”max –1

and its attributes are recorded. The complement to 1367.63 is then calculat-ed, becoming the new anchor ion (AI) and the process continues.

“virtual” b1, 100.0625

b2

Starting Extend by One from y” max

0

WORKFLOWS

1

2

2 3 3

1

2

4

Compliment Pairs

Extension m/z (AA1-j)

Table 1a Table 1b

Table 1c

1

2

3

3

4

5

5

4 4

5

b5

Figure 6a & 6b Illustrate respectively the post RDIF processing product ions in Table 3 by Ion and Fragment Type. Figure 6b illustrates compliment pairs (red) the number associated to each line connections refers to the m/z pair illustrated in Table 1b. The blue lines represent the product ions by exten-sion. Figure 6b illustrates these same product ions by fragment type (y”, b) also illustrated are the “virtual” by ex-tension product ions

Given Post precursor and product ion alignment, we have a list of precursor- product ions comprising: Precursor isotope charge cluster at charge state zpc and monoisotopic mass A0 (m/zpc)

Precursor Residual monoisotopic mass A0; i.e., unfragmented precursor found in EE (if found)

Filtered Product Ion spectra: list of EE product ions filtered by intensity and m/z

Anchor Ion table pre-populated with calculated values for: y”max, bmax, y”1 (K), y”1

(R)

Find Anchor Ions 1. Search the product ions in the Filtered Product Ion Table to find anchor ions: 1.1 If a match is found, then: 1.1.1 Mark this ion as ‘matched’

Find Complementary Pairs 1. Calculate the precursor protonated mass: [Mr,pc+zpcH]z+ = (m/zpc– mp) * zpc + z * mp

where Mr,pc is the relative molecular mass of the precursor, m/zpc is the monoisotopic mass of the precursor charge cluster A0 isotope, zpc is the charge state of the precursor, mp is the mass of a proton

2. For each ion (i) in the filtered product spectrum, do: 2.1 For each possible product ion charge state: zp = 1,2,…,(zpc-1), do:

2.1.1 Compute the product ion (i) protonated mass: [Mr,i+ziH]z+ at charge state zp [Mr,i+ziH]z+ = m/zi * zp

2.1.2 Compute the complementary ion protonated mass [Mr,c+zcH]z+ from the precursor: [Mr,c+zcH]z+ = [Mr,pc+zpcH]z+ - [Mr,i+ziH]z+ where Mr,c is the relative molecular mass of the complementary product ion and Mr,c + Mr,i = Mr,pc by definition.

2.1.3 Reduce the complementary protonated mass [Mr,c+zcH]z+ to the target ion mass m/zc at the complementary charge state zc = zpc - zp: m/zc = [Mr,c+zcH]z+ / zc

2.1.4 Search all the other ions in the filtered product spectrum for a complementary match to m/zc (within +/- ppm tolerance): 2.1.4.1 If a match is found, then

2.1.4.1.1 annotate the charge state of this ion: z zc 2.1.4.1.2 mark it as ‘matched’ 2.1.4.1.3 add it to the list of anchor ions.

2.1.4.1.4 Stop the search.

Identify Remaining Product Ions—Use Anchors & C-Pairs to Extend Peptide Sequence by 1 Amino Acid 1. For each anchor ion AIi in the Anchor Ion Table (includes Complements & on-the-fly “Virtuals”),

starting in the order of: y”max, bmax, y”1 (K), y”1

(R) do: 1.1. For each amino acid AAj in the Amino Acid Table (Table 1c.) do:

1.1.1 Start a new sequence chain n: (denotes a particular precursor/sequence) 1.1.2 Set direction sense: -1 for y-type, +1 for b-type

1.1.3 Calculate extend-by-one target mass m/zT: m/zT = m/zi + direction * m/zj / zi

1.1.4 Search unmatched filtered product ions for a target match (+/- ppm tolerance): 1.1.4.1 If a match is found, then:

1.1.4.1.1 Annotate this product ion’s properties accordingly: z zi (charge state of the anchor ion) Frag type same as AIi

AA type same as AAj chain number n (relates to the number of AAj matching in one loop)

1.1.4.1.2 Mark this ion as matched 1.1.4.1.3 Calculate the Complement and look for a match 1.1.4.1.4 If a match is found, then:

1.1.4.1.4.1 Annotate ion as per 1.1.4.1.1 Else 1.1.4.1.4.2 Create on-the-fly “virtual” 1.1.4.1.4.3 Make AIi = on-the-fly “virtual” (m/zVC) 1.1.4.1.4.4 switch Ion Type (from y” to b) 1.1.4.1.4.5 Continue to step 1

1.1.2.2 At completion, IF Match Count = 0 then, Else GoTo 1. Create “virtual” where m/zT = m/zVT = m/zi - m/zj / zi and that becomes

the new anchor) 2. Transfer ‘matched’ product ions from step 1 to Final Product Ion Table 3. For each product ion in Final Product Ion Table, do:

3.1 Query for isotopes using A0 intensity, z, and delta m/z 3.2 Check Glyco. Status

3.2.1 If Glyco Status = “yes”, then 1. where AAj = Glyco Table (Table 3) 3.3 Check Isobaric Labels (TMT, iTRAQ)

3.3.1 If a match is found, then 3.3.1.1 Get reference m/z and Quantify

3.4 Check SILAC Status 3.4.1 If SILAC Status = “yes”, then 3.4.1.1 Match SILAC pairs 3.4.1.2 Annotate ion as SILAC pair per 1.1.4.1. 3.4.1.3 Quantify

CONCLUSION

Most if not all DIA Product Ion Spectra are Chimeric

Product Ions to Co-Eluting/Co-Fragmenting Precursors can be Identified and Correctly Quantified provided some continuity in their fragmentation patterns exist

Product Ion Mass Accuracy is paramount in grouping product ions to their respective parent precursor in chimeric product ion spectra

Often it is a plurality of peptides that best represent a composite DIA product ion spectra

Figure 3.

Figure 2b.

Figure 2a.

The six most intense precursor ions illustrated in Figure 1 were processed using the described workflow. Figures 2a & 2b illus-trate flow diagrams relating to respectively, initial product ion filtering and complementary pair generation. Figure 3 repre-sents another flow diagram describing the process of extend-ing Anchor Ion’s (y”max & bmax, Compliments, y”1, Virtual & Vir-tual Complements) one m/z at a time. A m/z can be any user-defined value. As examples Table 1c illustrates the typical m/z values for tryptic peptide where Table 2 illustrates the those used in calculating glycan structures. With respect to quantification the described workflow as illustrated can quanti-fy samples utilizing either isobaric (TMT/iTRAQ) or isotopic SI-LAC labelled samples. For TMT/iTRAQ the user provides the reference product ion m/z in the initial setup.

Table 2

Figure 4A-D illustrate the initial process of enhancing precursor and product ion alignment. To begin, the mass analyzer detected 8331 product ions above the LOD. Filtering by m/z and Intensity (<y”max, < Intensity/250) reduced the count to 371 (4.2%), compliment pairs to 32 Ions, 16 complement pairs.

Table 3 References

1. Geromanos, S. J., Hughes, C., Golick, D., Ciavarini, S., Gorenstein, M. V., Richardson, K., Hoyes, J. B., Vissers, J. P.C. and Langridge, J. I. (2011), Simulating and validating proteomics data and search results. Proteomics, 11: 1189–1211. doi: 10.1002/pmic.201000576 2. Geromanos SJ, Hughes C, Ciavarini S, Vissers JP, Langridge JI. Using ion purity scores for enhancing quantitative accuracy and precision in complex proteomics samples. Anal Bioanal Chem. 2012 Sep;404 (4):1127-39. doi:10.1007/s00216-012-6197-y. Epub 2012 Jul 19. PubMed PMID: 22811061. 3. Senko et al, J Am Soc MS 1995 pp. 229-233

Two of a series TP-156, WP-325, MP-435, TP-544

599.3190, 2+

599.3190, 2+

790.3990, 2+

484.7469, 2+

Figure 6a

Figure 6a

Figure7A

B

C

y”9

y”8 5x

*

y”1

y”1

y”10

y”9

*

In Figure 1A, 9 of the top 10 precursor ions (by intensity) were iden-tified by RDIF processing. Due to space limitations presented is a high, middle and low intensity precursor. ~33% resulted in more than one peptide assignment. Figure 7 panels A & B represent 2-chains off the 599.3190, 2+ precursor. Panel A, illustrates a peptide from G3P1_YEAST, Mr 1196.6190. RDIF processing uncovered a sec-ond chain, Panel B, identifying a second peptide from GID3_YEAST, Mr 1196.6077. (~10ppm). 240K mass resolving power would be re-quired to separate both at the baseline. The variation in fragment intensity of the y”7 & y”8 ions in Panel B relate to the 799.3985 ion also being the b7 (Table 3) of the 790.3990, from YEAST_PGK while y”8 is also the 1+ of the 484.7469, 2+ from YEAST_ADH1.

y”6 (X-P)

y”7

y”6

y”5

y”4

y”3 y”2

y”1

b1 b2 b3 b5 b4 b7

b6

b1

b2

b3

b5

b8 b7 b6 b4

b9

b1

b2

b3

b5

b4 b6

b8 b7 b9

y”4

y”3

y”2 y”4

y”5 y”6

** y”7